Real-time PCR point mutation assays for detecting HIV-1 resistance to antiviral drugs

ABSTRACT

Disclosed are compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids, such as the nucleic acids encoding the reverse transcriptase or protease of HIV as disclosed herein. Thus, provided is an oligonucleotide comprising any one of the nucleotide sequences set for in SEQ ID NOS:1-89, and 96-104. Also provided are the oligonucleotides consisting of the nucleotides as set forth in SEQ ID NOS:1-89, and 96-104. Each of the disclosed oligonucleotides is a probe or a primer. Also provided are mixtures of primers and probes and for use in RT-PCR and primary PCR reactions disclosed herein. Provided are methods for the specific detection of several mutations in HIV. Mutations in both the reverse transcriptase and the protease of HIV can be detected using the methods described herein.

CROSS REFERENCE TO RELATED PATENT APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/602,366, filed Sep. 4, 2012, which is a divisional of U.S. patentapplication Ser. No. 13/253,463, filed Jan. 24, 2012 (now U.S. Pat. No.8,318,428), which is a continuation of U.S. patent application Ser. No.11/570,138 filed Dec. 7, 2006 (now U.S. Pat. No. 8,043,809), whichclaims priority to PCT/US2005/019907 filed Jun. 7, 2005, which claimspriority to U.S. provisional application Ser. No. 60/577,696 filed Jun.7, 2004, each of which is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

The HIV pandemic now exceeds 40 million persons and its expansion isbeing met with an increased use of anti-HIV drugs to care for the livesof those affected. Emergence of drug resistance is expected to increaseas the use of these drugs for the clinical management of HIV-1 infectedpersons increases worldwide. Highly active antiretroviral therapy(HAART) containing a combination of three antiretroviral drugs iscurrently recommended and has been effective in reducing mortality andmorbidity. Four classes of drugs are available that inhibit eithervirion entry (e.g., T-20), nucleotide extension by viral reversetranscriptase (e.g., 3TC, d4T), reverse transcriptase enzymatic activity(e.g., nevirapine, efavirenz), or the viral protease (e.g., nelfinavir,lopinavir). Drug resistance that is conferred by mutations is frequentlyselected in viruses from patients failing antiretroviral therapy and isconsidered a major cause of treatment failure.

Current treatment guidelines recommend baseline drug resistance testingfor the selection of optimal drug regimens for patients initiatingantiretroviral therapy. Accurate identification of any resistant virusesthe person carries will help guide the selection of treatment regimenswith fully active drugs. Drug resistance testing is performed throughthe use of phenotypic or genotypic assays. Phenotypic assays measuredrug susceptibilities of patient-derived viruses and provide directevidence of drug resistance. However, phenotypic assays areculture-based, complex, laborious, and costly. Genotypic assays arefrequently used to detect mutations associated with drug resistance bysequence analysis of the viral RNA from plasma. These assays are alsocomplex and are insensitive to the detection of low levels of mutants,such as what might be present early in the emergence of resistance orwhich might persist at low set points in the absence of treatment.Commonly-used sequencing methods do not reliably detect mutants presentat levels below 20-30% of the total viral population within a sample.Described in this application are PCR-based drug resistance detectionassays that are able to detect drug-resistant viruses present atfrequencies as low as 0.5%-0.04% within the plasma of infected persons.These sensitivities are 40-500-times greater than what has been achievedby conventional sequence testing.

Although drug resistance is frequently seen in patients failingantiretroviral therapy, a substantial prevalence (˜8-25%) of transmitteddrug-resistant HIV-1 is found among drug-naïve populations, supportingthe need for baseline drug resistant testing. Because drug-resistantmutants are generally less fit than wild type viruses in the absence ofdrug, many drug-resistance mutations revert back to wildtype over timeand become gradually undetectable in plasma. However, the drug-resistantviruses that become undetectable in plasma remain archived in thepatients and are re-selected when drugs are used. Therefore, it isimportant to have sensitive assays that can accurately detect thepresence of low frequency drug-resistant mutants. Data from the use ofthe sensitive real-time PCR assays described in this patent applicationdemonstrate clearly that conventional sequencing of drug-naïve personsunderestimates the prevalence of transmitted drug resistance (Johnson etal., 13th HDR Workshop, Tenerife, Spain, 2004). Testing transmitted drugresistant viruses for additional mutations by the sensitive assaysidentified new mutants that increased the prevalence of resistancewithin the population by another 2 to 8%. The increases imply that drugresistance mutations are transmitted at frequencies 20-80% higher thanpreviously reported. Therefore, these data demonstrate the poorersensitivity of sequencing methods for baseline drug resistance testing.

Drug resistance testing is also indicated for patients receiving HAARTto manage treatment failures and to help guide the selection of newHAART regimens with active drugs. Recent data have pointed to theimportance of sensitive drug resistance assays for this testing andassociate low-frequency drug-resistant viruses that are not detectableby conventional sequencing with poor treatment outcomes (Mellors et al.,11th CROI, 2004; Jourdain et al., JID 2004) (I). These studies reportedthat persons exposed to a non-nucleoside reverse transcriptase inhibitor(NNRTI) who generated resistance mutations detectable only by sensitiveassays, and not by conventional sequencing, respond more poorly tosubsequent NNRTI-containing regimens. Data from the subtype C HIV-1assays reported herein show that more than one-third of thedrug-resistant viruses that emerge from intrapartum single-dosenevirapine intervention are not identified by conventional populationsequencing (Johnson et al., 12th CROI 2005). The detection of thesubstantial numbers of low-frequency drug-resistant viruses will beimportant for selecting a regimen with fully active drugs.

In clinical monitoring of treated persons, the greater sensitivity ofthe present real-time PCR resistance assays over conventional sequencingmay allow earlier detection of resistance mutations that emerge duringtreatment and provide advance notice of possible declines in response totherapy. Early detection will help guide clinicians in modifying drugregimens in an effort to prevent treatment failure and the emergence ofhigh-level drug resistance. Methods with greater sensitivity indetecting low levels of resistant virus, below what is capable byconventional sequence analysis, are important for improving clinicalmanagement of patients under HAART. The substantially highersensitivity, the simplicity, the high throughput capability, and the lowcost of the present real-time PCR drug resistance assays are alladvantages over conventional sequence analysis.

SUMMARY OF THE INVENTION

Disclosed are compositions including primers and probes, which arecapable of interacting with the disclosed nucleic acids, such as thenucleic acids encoding the reverse transcriptase or protease of HIV asdisclosed herein. Thus, provided is an oligonucleotide comprising anyone of the nucleotide sequences set forth in SEQ ID NOS:1-89, and96-104. Also provided is an oligonucleotide consisting of any of thenucleotide sequences set forth in SEQ ID NOS:1-89, and 96-104. Each ofthe disclosed oligonucleotides is a probe or a primer. Also provided aremixtures of primers and mixtures of primers and probes and for use inRT-PCR and primary PCR reactions disclosed herein. Kits comprising theprimers or probes are provided. Provided are methods for the specificdetection of several mutations in HIV. Mutations in both the reversetranscriptase and the protease of HIV can be detected using the methodsdescribed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are schematic illustrations of the principle of thepresent assay.

FIG. 2A shows the sensitivity of the assay for 184V.

FIG. 2B shows the lower limit for 184V detection in clinical specimens.

FIG. 3 shows the performance of the 184V assay on clinical specimens.

FIG. 4 shows the ΔCT frequency distribution for clinical samples.

FIG. 5 shows the ΔCT values for real-time PCR analysis of the 103Nmutation in pre-NVP and post-NVP plasma samples.

DETAILED DESCRIPTION OF THE INVENTION

In an effort to improve the detection of mutations associated with HIV-1drug resistance, provided are PCR-based point mutation assays. Thepresent methodology allows testing for different point mutations inpatient samples at an achievable sensitivity of 1-2 log greater thanconventional sequencing. The principle of the present assay is tocompare the differential amplifications of a mutation-specific PCR and atotal copy (common) PCR, which detects all sequences present. The assaycan use template generated from RT-PCR of viral RNA or from PCR ofproviral DNA from infected cells (FIG. 1).

Two important HIV-1 reverse transcriptase mutations that significantlycompromise the success of treatment with reverse transcriptaseinhibitors are 103N and 184V. The 103N mutation is frequently selectedin patients failing treatment with non-nucleoside RT inhibitors (e.g.,nevirapine, efavirenz). Likewise, the frequent appearance of the 184Vmutation following exposure to nucleoside inhibitors lamivudine (3TC)and abacavir, and it's seemingly rapid disappearance afterdiscontinuation of therapy, makes accurate measure of these mutationsimportant for surveillance and clinical management.

The simplicity, greater sensitivity, and high-throughput capabilities ofthe present real-time PCR methodology make it useful for screening largenumbers of samples, which allows the implementation of universalresistance testing and protracted surveillance of resistance mutations.

The methods disclosed herein have multiple applications including (1)resistance testing for clinical management of HIV-infected personsreceiving anti-HIV drugs (for detecting emergence of resistant virusesin treated persons, and as a pre-treatment evaluation of patientbaseline HIV in order to tailor the most appropriate drug combination),(2) use in blood bank screening as a nucleic acid test (NAT), due to thehigh sensitivity and high throughput capability of the assays, (3) theability to measure plasma viral loads, since the assays are inherentlyquantitative, (4) use as a screening tool for monitoring the spread ofresistant HIV, (5) use as a research tool to study the emergence andbiology of drug resistance mutations, (6) detection of resistancemutations in both subtype B and non-B subtypes of HIV-1, (7) detectionof resistance mutations in HIV-2, and (8) identification of specificpanels of mutations that are designed to address each of the describeduses. The reagents and specific usages developed here are unique.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a primer” includesmixtures of two or more such primers, and the like.

Compositions

Disclosed are compositions including primers and probes, which arecapable of interacting with the disclosed nucleic acids, such as thenucleic acids encoding the reverse transcriptase or protease of HIV asdisclosed herein and in the literature.

Thus, provided is an oligonucleotide comprising a nucleotide sequence asset forth in any of SEQ ID NOS:1-89, and 96-104. Also provided is anoligonucleotide consisting of any one of the nucleotide sequences setforth in SEQ ID NOS: 1-89, and 96-104. Thus, provided is anoligonucleotide comprising the sequence selected from the groupconsisting of the nucleotides as set forth in the sequence listing asSEQ ID NOS: 1-89, and 96-104. Each of the disclosed oligonucleotides isa probe or a primer. Each can be used independently of the others in anamplification method or in a hybridization/probing method. One or moreof the probes or primers can be used together in the compositions andmethods for detecting mutations. Specific examples of such compositionsand methods are described herein.

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Anon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate). The term“nucleotide” includes nucleotides and nucleotide analogs, preferablygroups of nucleotides comprising oligonucleotides, and refers to anycompound containing a heterocyclic compound bound to a phosphorylatedsugar by an N-glycosyl link or any monomer capable of complementary basepairing or any polymer capable of hybridizing to an oligonucleotide.

The term “nucleotide analog” refers to molecules that can be used inplace of naturally occurring bases in nucleic acid synthesis andprocessing, preferably enzymatic as well as chemical synthesis andprocessing, particularly modified nucleotides capable of base pairing. Anucleotide analog is a nucleotide which contains some type ofmodification to one of the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties. This term includes, but is not limitedto, modified purines and pyrimidines, minor bases, convertiblenucleosides, structural analogs of purines and pyrimidines, labeled,derivatized and modified nucleosides and nucleotides, conjugatednucleosides and nucleotides, sequence modifiers, terminus modifiers,spacer modifiers, and nucleotides with backbone modifications,including, but not limited to, ribose-modified nucleotides,phosphoramidates, phosphorothioates, phosphonamidites, methylphosphonates, methyl phosphoramidites, methyl phosphonamidites,5′-β-cyanoethyl phosphoramidites, methylenephosphonates,phosphorodithioates, peptide nucleic acids, achiral and neutralinternucleotidic linkages and normucleotide bridges such as polyethyleneglycol, aromatic polyamides and lipids. Optionally, nucleotide analog isa synthetic base that does not comprise adenine, guanine, cytosine,thymidine, uracil or minor bases. These and other nucleotide andnucleoside derivatives, analogs and backbone modifications are known inthe art (e.g., Piccirilli J. A. et al. (1990) Nature 343:33-37; Sanghviet al (1993) In: Nucleosides and Nucleotides as Antitumor and AntiviralAgents, (Eds. C. K. Chu and D. C. Baker) Plenum, N.Y., pp. 311-323;Goodchild J. (1990) Bioconjugate Chemistry 1:165-187; Beaucage et al.(1993) Tetrahedron 49:1925-1963).

Nucleotide substitutes include molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

There are a variety of molecules disclosed herein that are nucleic acidbased. The disclosed nucleic acids are made up of for example,nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limitingexamples of these and other molecules are discussed herein.

The term “oligonucleotide” means a naturally occurring or syntheticpolymer of nucleotides, preferably a polymer comprising at least threenucleotides and more preferably a polymer capable of hybridization.Oligonucleotides may be single-stranded, double-stranded, partiallysingle-stranded or partially double-stranded ribonucleic ordeoxyribonucleic acids, including selected nucleic acid sequences,heteroduplexes, chimeric and hybridized nucleotides and oligonucleotidesconjugated to one or more nonoligonucleotide molecules. In general, thenucleotides comprising a oligonucleotide are naturally occurringdeoxyribonucleotides, such as adenine, cytosine, guanine or thyminelinked to 2′-deoxyribose, or ribonucleotides such as adenine, cytosine,guanine or uracil linked to ribose. However, an oligonucleotide also cancontain nucleotide analogs, including non-naturally occurring syntheticnucleotides or modified naturally occurring nucleotides. Such nucleotideanalogs are well known in the art and commercially available, as arepolynucleotides containing such nucleotide analogs (Lin et al., Nucl.Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73(1997), each of which is incorporated herein by reference).

The term “polynucleotide” is used broadly herein to mean a sequence oftwo or more deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “polynucleotide”includes RNA and DNA, which can be a gene or a portion thereof, a cDNA,a synthetic polydeoxyribonucleic acid sequence, or the like, and can besingle stranded or double stranded, as well as a DNA/RNA hybrid.Furthermore, the term “polynucleotide” as used herein includes naturallyoccurring nucleic acid molecules, which can be isolated from a cell, aswell as synthetic molecules, which can be prepared, for example, bymethods of chemical synthesis or by enzymatic methods such as by thepolymerase chain reaction (PCR). In various embodiments, apolynucleotide of the invention can contain nucleoside or nucleotideanalogs, or a backbone bond other than a phosphodiester bond. Ingeneral, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can contain nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73(1997), each of which is incorporated herein by reference).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22:977-986 (1994); Eckerand Crooke, BioTechnology 13:351360 (1995), each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation. Functional analogs of naturally occurring polynucleotidescan bind to RNA or DNA, and include peptide nucleic acid (PNA)molecules.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art which do not interfere withthe enzymatic manipulation.

The oligonucleotides of SEQ ID NOS:1-89, and 96-104 can be modified ininsubstantial ways and yet retain substantially the same hybridizationstrength and specificity as described herein. These parameters areeasily measured in assays such as those taught herein. Thus, one ofskill in the art will be able to envision a number of nucleotidesubstitutions to the disclosed sequences, so long as they retain 80%sequence similarity with the specifically disclosed sequence. Primersand probes of the invention can include sequences having at least 85%,90%, 95%, 96%, 97%, 98% or 99% similarity to one of SEQ ID NOS:1-89, and96-104 are envisioned. More specifically, primers and probes withsubstitutions based on known sequences of the HIV-1 protease or reversetranscriptase are envisioned because these alternative sequences areenvisioned by the person of skill in this art.

In certain embodiments the primers are used to support DNA amplificationreactions. Typically the primers are capable of being extended in asequence specific manner. Extension of a primer in a sequence specificmanner includes any methods wherein the sequence and/or composition ofthe nucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner. Typically thedisclosed primers hybridize with the disclosed nucleic acids or regionof the nucleic acids or they hybridize with the complement of thenucleic acids or complement of a region of the nucleic acids.

The oligonucleotides described herein include primers and probeseffective for cross subtype reactive PCR, as such, they are capable ofdetecting mutations in a variety of HIV subtypes. The following primersand probes can also include additions known to those skilled in the art.Examples of such additions include, but are not limited to, moleculesfor linking the primer to a substrate, and the like. Furthermore, ifdesired, a nucleic acid molecule of the invention can incorporate adetectable moiety. As used herein, the term “detectable moiety” isintended to mean any suitable label, including, but not limited to,enzymes, fluorophores, biotin, chromophores, radioisotopes, coloredparticles, electrochemical, chemical-modifying or chemiluminescentmoieties. Examples include (i) enzymes which can catalyze color or lightemitting (luminescence) reactions and (ii) fluorophores. The detectionof the detectable moiety can be direct provided that the detectablemoiety is itself detectable, such as, for example, in the case offluorophores. Alternatively, the detection of the detectable moiety canbe indirect. In the latter case, a second moiety reactable with thedetectable moiety, itself being directly detectable is preferablyemployed. The detectable moiety may be inherent to a molecular probe.Common fluorescent moieties include: fluorescein, cyanine dyes,coumarins, phycoerythrin, phycobiliproteins, dansyl chloride, Texas Red,and lanthanide complexes.

Provided are the following: An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:1; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:2. An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO: 3; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO: 4;An oligonucleotide comprising the nucleotides as set forth in SEQ ID NO:5; An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO: 6; An oligonucleotide comprising the nucleotides as set forth in SEQID NO: 7; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO: 8; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO: 9; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:10. An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:11; An oligonucleotide comprising the nucleotidesas set forth in SEQ ID NO:12; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:13; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:14; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:15; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:16;An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:17; An oligonucleotide comprising the nucleotides as set forth in SEQID NO:18; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:19; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:20; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:21; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:22; An oligonucleotide comprising the nucleotidesas set forth in SEQ ID NO:23; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO: 24; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO: 25; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO: 26; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:27; An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO: 28; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO: 29; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO: 30; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:31; An oligonucleotide comprising the nucleotidesas set forth in SEQ ID NO:32; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:33; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:34; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:35; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:36; An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:37; An oligonucleotide comprising the nucleotides as set forth in SEQID NO:38; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:39; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:40; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:41; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:42; An oligonucleotide comprising the nucleotidesas set forth in SEQ ID NO:43; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:44; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:45; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:46. Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:47;An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:48; An oligonucleotide comprising the nucleotides as set forth in SEQID NO:49; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:50; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:51; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:52; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:53; An oligonucleotide comprising the nucleotidesas set forth in SEQ ID NO:54; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:55; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:56; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:57; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:58;An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:59; An oligonucleotide comprising the nucleotides as set forth in SEQID NO:60; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:61; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:62; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO: 63; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO: 64; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:65; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:66; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:67; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:68;An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:69; An oligonucleotide comprising the nucleotides as set forth in SEQID NO:70; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:71; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:72; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:73; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:74; An oligonucleotide comprising the nucleotidesas set forth in SEQ ID NO:75; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:76; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:77; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:78; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:79;An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:80; An oligonucleotide comprising the nucleotides as set forth in SEQID NO:81; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:82; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:83. An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:84; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO: 85; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:86; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:87; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:88; Anoligonucleotide comprising the nucleotides as set forth in SEQ ID NO:89;An oligonucleotide comprising the nucleotides as set forth in SEQ IDNO:96; An oligonucleotide comprising the nucleotides as set forth in SEQID NO: 97; An oligonucleotide comprising the nucleotides as set forth inSEQ ID NO:98; An oligonucleotide comprising the nucleotides as set forthin SEQ ID NO:99; An oligonucleotide comprising the nucleotides as setforth in SEQ ID NO:100; An oligonucleotide comprising the nucleotides asset forth in SEQ ID NO:101; An oligonucleotide comprising thenucleotides as set forth in SEQ ID NO:102; An oligonucleotide comprisingthe nucleotides as set forth in SEQ ID NO:103; An oligonucleotidecomprising the nucleotides as set forth in SEQ ID NO:104. Thus, providedis an oligonucleotide comprising the sequence selected from groupconsisting of the nucleotides as set forth in the sequence listing asSEQ ID NO:1-89, and 96-104.

Also provided are mixtures of primers for use in RT-PCR and primary PCRreactions disclosed herein. Thus, a mixture of primers comprising SEQ IDNO:1 and 3 is provided. This mixture can be used for the reversetranscription-PCR (RT-PCR) reaction and the primary PCR reaction forHIV. It reverse transcribes and amplifies the HIV protease regioncomprising positions 30 and 90 in addition to the region of the reversetranscriptase gene comprising the mutations described herein.

Provided is a mixture of primers comprising SEQ ID NOS:2 and 3. Thismixture does not reverse transcribe or amplify the protease regions ofinterest, but is useful for the analysis of the reverse transcriptase.

A mixture of primers comprising SEQ ID NOS:4 and 6 is provided. Thismixture is for the RT-PCR and primary PCR reactions for HIV. It alsoreverse transcribes and amplifies the HIV protease region comprisingpositions 30 and 90 in addition to the region of the reversetranscriptase gene comprising the mutations described herein.

Also provided is a mixture of primers comprising SEQ ID NOS:5 and 6.This mixture does not reverse transcribe or amplify the protease regionsof interest, but is useful for the analysis of the reversetranscriptase.

Provided are oligonucleotide mixtures for use in the mutation-specificPCR reactions disclosed herein. Detection can be achieved so long as anyof the disclosed forward primers are paired with any of the reverseprimers for a given mutation.

Thus, provided is a mixture of primers comprising one or more primersselected from the group consisting of SEQ ID NOS:22,23,24 and 25. Thisis a forward primer mixture for the 103N mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer consisting of SEQ ID NO:26.

Also provided is a mixture of primers comprising one or more primersselected from the group consisting of SEQ ID NOS: 59,60 and 61. This isa forward primer mixture for the 103N mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer consisting of SEQ ID NO:26.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:33,34 and 35 is provided. This is aforward primer mixture for the 184V mutation-specific PCR reaction. Themixture can further include a reverse primer. For example, the reverseprimer can be a primer comprising or consisting of SEQ ID NO:36.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:88, 89, 102, 103, and 104 is provided.This is a forward primer mixture for the 184V mutation-specific PCRreaction. The mixture can further include a reverse primer. For example,the reverse primer can be a primer comprising or consisting of SEQ IDNO:85.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:62,63,64,65,96 and 97 is provided. Thisis a forward primer mixture for the 41L mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:66.

A mixture of primers comprising SEQ ID NOS:10 and 98 and a reverseprimer is provided. This mixture includes a forward primer for the 65Rmutation-specific PCR reaction. The reverse primer can, for example, bea primer comprising or consisting of SEQ ID NO:11.

A mixture of primers comprising SEQ ID NOS:69 and 70 is provided. Thisis a forward primer mixture for the 67N mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:8.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:12,13, and 71 is provided. This is aforward primer mixture for the 69T mutation-specific PCR reaction. Themixture can further include a reverse primer. For example, the reverseprimer can be a primer comprising or consisting of SEQ ID NOS:8 and 14.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:2,16,17,18,19, and 100 is provided. Thisis a forward primer mixture for the 70R mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer comprising or consisting of SEQ IDNOS:20,72, or 73.

A mixture of primers comprising SEQ ID NOS:28 and 29 is provided. Thisis a forward primer mixture for the 181C mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:30.

A mixture of primers comprising SEQ ID NOS:83 and 84 is provided. Thisis a forward primer mixture for the protease 181C mutation-specific PCRreaction. The mixture can further include a reverse primer. For example,the reverse primer can be a primer comprising or consisting of SEQ IDNO:85.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:38,39,74,75, and 101 is provided. This isa forward primer mixture for the 215T mutation-specific PCR reaction.The mixture can further include a reverse primer. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:45.

A mixture of primers comprising SEQ ID NO:40 and a reverse primer isprovided. This is a primer mixture for the 215Y mutation-specific PCRreaction. The reverse primer can be for example, a primer comprising orconsisting of SEQ ID NO:45.

A mixture of primers comprising SEQ ID NO:41 and a reverse primer isprovided. This is a primer mixture for the 215F mutation-specific PCRreaction. The reverse primer can be, for example, a primer comprising orconsisting of SEQ ID NO:45.

A mixture of primers comprising SEQ ID NO:42 and a reverse primer isprovided. This is a primer mixture for the 215S mutation-specific PCRreaction. The reverse primer can, for example, be a primer comprising orconsisting of SEQ ID NO:45.

A mixture of primers comprising SEQ ID NO:43 and a reverse primer isprovided. This is a primer mixture for the 215C mutation-specific PCRreaction. The reverse primer can, for example, be a primer comprising orconsisting of SEQ ID NO:45.

A mixture of primers comprising SEQ ID NO:44 and a reverse primer isprovided. This is a primer mixture for the 215D mutation-specific PCRreaction. The reverse primer can, for example, be a primer comprising orconsisting of SEQ ID NO:45.

A mixture of primers comprising SEQ ID NOS:48 and 49 is provided. Thisis a forward primer mixture for the protease 30N mutation-specific PCRreaction. The mixture can further include a reverse primer. For example,the reverse primer can be a primer comprising or consisting of SEQ IDNO:50.

A mixture of primers comprising one or more primers selected from thegroup consisting of SEQ ID NOS:53,54,55,78,79 and 80 is provided. Thisis a forward primer mixture for the protease 90M mutation-specific PCRreaction. The mixture can further include a reverse primer. For example,the reverse primer can be a primer comprising or consisting of SEQ IDNOS:56 and 81.

Also provided are mixtures of primers for mutation-specific PCR reactionfor reverse transcriptase and protease. These mixtures can comprise aforward and reverse primer for a reverse transcriptase mutation and aforward and reverse primer for a protease mutation. The forward primersin the mixture can include any forward primer for the specific RTmutation to be detected and any forward primer for the protease mutationto be detected. These mixtures can be used to simultaneously detect bothan RT mutation and a protease mutation. An example of such a mixture ofprimers comprises or consists of SEQ ID NOS: 33,34, 35, 78,79, and 80.This is a forward primer mixture for the reverse transcriptase 184V andthe 90M protease mutations. The mixture can further include reverseprimers. For example, the reverse primers can comprise or consist of SEQID NOS: 36 and 81.

The mixtures (and methods) disclosed herein can utilize reverse primersother than those exemplified. The exemplified reverse primers were foundto work well. However, the requirements of the reverse primer in thepresent method are typical of reverse primers designed and usedroutinely, and other reverse primers can be routinely made and used. Itis expected that the reverse primer will be within about 40 to 250 basesfrom the forward primer. It is also expected that the reverse primerwill be positioned in a stable location lacking a degree of variabilitythat would impede binding. The reverse primer is most likely to belocated in the RT gene or the protease gene, but the exact location isroutinely variable based on the usual criteria for reverse primerpositioning.

Amplification mixtures are provided that include a probe for use in areal time PCR reaction. The mixtures can thus include a forward primer,a reverse primer and a probe. For example, an amplification mixture isprovided comprising a forward primer or a mixture of forward primersthat amplifies the 103N, 65R, 69T and 70R mutations, wherein the mixturefurther comprises an oligonucleotide having the nucleotides as set forthin SEQ ID NO:9. This is an example of a probe that can be used in any ofthese mutation-specific PCR reactions. This probe can also be used inthe total copy PCR reaction.

An amplification mixture is provided comprising a forward primer or amixture of forward primers that amplifies the 41L mutations, wherein themixture further comprises an oligonucleotide having the nucleotides asset forth in SEQ ID NO:67. This is an example of a probe that can beused in mutation-specific PCR reactions for this mutation.

An amplification mixture is provided comprising a forward primer or amixture of forward primers that amplifies the 65R, 67N, and 69Tmutations, wherein the mixture further comprises an oligonucleotidehaving the nucleotides as set forth in SEQ ID NO:68. This is an exampleof a probe that can be used in any of these mutation-specific PCRreactions.

An amplification mixture is provided comprising a forward primer or amixture of forward primers that amplifies the 70R mutation, wherein themixture further comprises an oligonucleotide having the nucleotides asset forth in SEQ ID NOS:9 or 67. This is an example of a probe that canbe used in mutation-specific PCR reactions for this mutation.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the 181C and 184V mutations,wherein the mixture further comprises an oligonucleotide having thenucleotides as set forth in SEQ ID NO:32. This is an example of a probethat can be used in either of these mutation-specific PCR reactions.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the 215 mutations, wherein themixture further comprises an oligonucleotide having the nucleotides asset forth in SEQ ID NOS:47, 76, or 77. These are examples of probes thatcan be used in any of these mutation-specific PCR reactions.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the protease 30N mutation,wherein the mixture further comprises an oligonucleotide having thenucleotides as set forth in SEQ ID NO:52. This is an example of a probethat can be used in mutation-specific PCR reactions for this mutation.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the protease 90M mutation,wherein the mixture further comprises an oligonucleotide having thenucleotides as set forth in SEQ ID NOS:58 or 82. This is an example of aprobe that can be used in mutation-specific PCR reactions for thismutation.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the 103N mutation, wherein themixture further comprises an oligonucleotide having the nucleotides asset forth in SEQ ID NO: 9. This is an example of a probe that can beused in mutation-specific PCR reactions for this mutation.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the 181C mutation, wherein themixture further comprises an oligonucleotide having the nucleotides asset forth in SEQ ID NOS:86 or 87. These are examples of probes that canbe used in mutation-specific PCR reactions for this mutation.

An amplification mixture is provided comprising a forward primer ormixture of forward primers that amplifies the 184V mutation, wherein themixture further comprises an oligonucleotide having the nucleotides asset forth in SEQ ID NOS:86, or 87. These are examples of probes that canbe used in mutation-specific PCR reactions for this mutation.

The probe can incorporate a detectable moiety. As used herein, the term“detectable moiety” is intended to mean any suitable label, including,but not limited to, enzymes, fluorophores, biotin, chromophores,radioisotopes, colored particles, electrochemical, chemical-modifying orchemiluminescent moieties. Examples include (i) enzymes which cancatalyze color or light emitting (luminescence) reactions and (ii)fluorophores. The detection of the detectable moiety can be directprovided that the detectable moiety is itself detectable, such as, forexample, in the case of fluorophores. Alternatively, the detection ofthe detectable moiety can be indirect. In the latter case, a secondmoiety reactable with the detectable moiety, itself being directlydetectable is preferably employed. The detectable moiety may be inherentto a molecular probe. Common fluorescent moieties include: fluorescein,cyanine dyes, coumarins, phycoerythrin, phycobiliproteins, dansylchloride, Texas Red, and lanthanide complexes.

The size of the primers or probes for interaction with the nucleic acidscan be any size that supports the desired enzymatic manipulation of theprimer, such as DNA amplification or the simple hybridization of theprobe or primer. A typical primer or probe would be at least, less thanor equal to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,3500, or 4000 nucleotides long. Primers or probes of any length betweenthe specified numbers are specifically contemplated.

The primers for the reverse transcriptase gene or protease genetypically will be used to produce an amplified DNA product that containsa region of the reverse transcriptase gene or protease gene containingthe relevant site(s) of the mutation(s) of interest. In general,typically the size of the product will be such that the size can beaccurately determined to within 3, or 2 or 1 nucleotides. This productcan be at least, less than or equal to 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250,1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotideslong.

In the mixtures and methods described herein, the specific probesdescribed are merely examples. Applying routine skill to the teachingherein, the person in this field can envision and make additional probesthat will function in the PCR compositions and methods described.

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995, incorporated herein by reference).

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6,incorporated herein by reference) to purely synthetic methods, forexample, by the cyanoethyl phosphoramidite method using a Milligen orBeckman System 1 Plus DNA synthesizer (for example, Model 8700 automatedsynthesizer of Milligen-Biosearch, Burlington, Mass. or ABI Model 380B).Synthetic methods useful for making oligonucleotides are also describedby Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriesterand phosphite-triester methods, incorporated herein by reference), andNarang et al., Methods Enzymol., 65:610-620 (1980), incorporated hereinby reference, (phosphotriester method). Protein nucleic acid moleculescan be made using known methods such as those described by Nielsen etal., Bioconjug. Chem. 5:3-7 (1994), incorporated herein by reference.

Also disclosed herein are kits that are drawn to reagents that can beused in practicing the methods disclosed herein. The kits can includeany reagent or combination of reagents discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended. Specific guidance as to the components of the kits is providedherein, including buffers, primers and probes. For example, disclosed isa kit for detecting a mutation in the reverse transcriptase gene orprotease gene of HIV, comprising one or more of the oligonucleotides setforth in SEQ ID Nos:1-92.

For further general information, an example of coding sequences of anHIV-1 protease and an HIV-1 reverse transcriptase are provided below.Also provided are accession numbers for these and other HIV-1 proteaseand an HIV-1 reverse transcriptase coding sequences. Accession numbersfor amino acid sequences of the HIV-1 reverse transcriptase and theHIV-1 protease are also provided. This information, along with sequenceinformation on many more examples of HIV-1 protease and reversetranscriptase proteins and coding sequences, are in the art. As such,they constitute a part of the disclosure of the present application.

HIV-1 Subtype B Genome

Accession Number: NC 001802, K03455

HIV-1 Protease

Exemplary Sequence (SEQ ID NO: 90) 1cctcaggtca ctctttggca acgacccctc gtcacaataa agataggggg gcaactaaag 61gaagctctat tagatacagg agcagatgat acagtattag aagaaatgag tttgccagga 121agatggaaac caaaaatgat agggggaatt ggaggtttta tcaaagtaag acagtatgat 181cagatactca tagaaatctg tggacataaa gctataggta cagtattagt aggacctaca 241cctgtcaaca taattggaag aaatctgttg actcagattg gttgcacttt aaatttt

Genome Location: 1799.2095

Additional Similar Nucleotide Examples: Accession Numbers: U31398,AJ279618, AJ279682, AJ279683, AJ279684

Protein: Accession Number: NP_705926

HIV-1 Reverse Transcriptase

Exemplary Sequence (SEQ ID NO: 91) 1cccattagcc ctattgagac tgtaccagta aaattaaagc caggaatgga tggcccaaaa 61gttaaacaat ggccattgac agaagaaaaa ataaaagcat tagtagaaat ttgtacagag 121atggaaaagg aagggaaaat ttcaaaaatt gggcctgaaa atccatacaa tactccagta 181tttgccataa agaaaaaaga cagtactaaa tggagaaaat tagtagattt cagagaactt 241aataagagaa ctcaagactt ctgggaagtt caattaggaa taccacatcc cgcagggtta 301aaaaagaaaa aatcagtaac agtactggat gtgggtgatg catatttttc agttccctta 361gatgaagact tcaggaagta tactgcattt accataccta gtataaacaa tgagacacca 421gggattagat atcagtacaa tgtgcttcca cagggatgga aaggatcacc agcaatattc 481caaagtagca tgacaaaaat cttagagcct tttagaaaac aaaatccaga catagttatc 541tatcaataca tggatgattt gtatgtagga tctgacttag aaatagggca gcatagaaca 601aaaatagagg agctgagaca acatctgttg aggtggggac ttaccacacc agacaaaaaa 661catcagaaag aacctccatt cctttggatg ggttatgaac tccatcctga taaatggaca 721gtacagccta tagtgctgcc agaaaaagac agctggactg tcaatgacat acagaagtta 781gtggggaaat tgaattgggc aagtcagatt tacccaggga ttaaagtaag gcaattatgt 841aaactcctta gaggaaccaa agcactaaca gaagtaatac cactaacaga agaagcagag 901ctagaactgg cagaaaacag agagattcta aaagaaccag tacatggagt gtattatgac 961ccatcaaaag acttaatagc agaaatacag aagcaggggc aaggccaatg gacatatcaa 1021atttatcaag agccatttaa aaatctgaaa acaggaaaat atgcaagaat gaggggtgcc 1081cacactaatg atgtaaaaca attaacagag gcagtgcaaa aaataaccac agaaagcata 1141gtaatatggg gaaagactcc taaatttaaa ctgcccatac aaaaggaaac atgggaaaca 1201tggtggacag agtattggca agccacctgg attcctgagt gggagtttgt taatacccct 1261cccttagtga aattatggta ccagttagag aaagaaccca tagtaggagc agaaaccttc 1321tatgtagatg gggcagctaa cagggagact aaattaggaa aagcaggata tgttactaat 1381agaggaagac aaaaagttgt caccctaact gacacaacaa atcagaagac tgagttacaa 1441gcaatttatc tagctttgca ggattcggga ttagaagtaa acatagtaac agactcacaa 1501tatgcattag gaatcattca agcacaacca gatcaaagtg aatcagagtt agtcaatcaa 1561ataatagagc agttaataaa aaaggaaaag gtctatctgg catgggtacc agcacacaaa 1621ggaattggag gaaatgaaca agtagataaa ttagtcagtg ctggaatcag gaaagtacta

Genome Location: 2096 . . . 3775

Additional Similar Nucleotide Examples: Accession Numbers: U28646,U28647, U28648, U28649. U53870, U53871

Protein: Accession Number: NP_705927

HIV-1 Subtype C Genome

Accession Number: AY162225, AY158533, DQ011180, DQ01173, AY049710

HIV-1 Protease

Exemplary Sequence (SEQ ID NO: 92) 1cctcaaatca ctctttggca gcgacccctt gtcacaataa aagtaggggg tcagataaag 61gaggctctct tagatacagg agcagatgat acagtattag aagacataaa tttgccagga 121aaatggaaac caaaaatgat aggaggaatt ggaggtttta tcaaagtaag acagtatgat 181caaatactta tagaaatttg tggaaaaaag gctataggta cagtattagt gggacccaca 241cctgtcaaca taattggaag aaatatgttg actcagcttg gatgcacact aaatttt

Genome Location: 2215 . . . 2511

Additional Similar Nucleotide Examples: Accession Numbers: AY510039,AY510043, AY589869

Protein: Accession Number: AAR92431

HIV-1 Reverse Transcriptase

Exemplary Sequence (SEQ ID NO: 93) 1CCAATTAGTC CYATTGAAAC TGTACCAGTA AAATTAAAGC CAGGGATGGA TGGCCCAAAG 61GTCAAACAAT GGCCATTGAC AGAAGAAAAA ATAAAAGCAT TAATAGCAAT TTGTGAAGAG 121ATGGAGAAGG AAGGAAAAAT TACAAAAATT GGGCCTGAAA ATCCATATAA CACCCCAGTA 181TTTGCCATAA AAAAGAAGGA CAGTACTAAG TGGAGAAAAT TAGTAGATTT CAGGGAACTC 241AATAAAAGAA CTCAAGACTT TTGGGAAGTT CAATTAGGGA TACCACACCC AGCAGGGTTA 301AAGAAAAAGA AATCAGTAAC AGTACTGGAT GTGGGGGATG CATATTTTTC AGTTCCTTTA 361GATAAAGACT TCAGAAAATA TACTGCATTC ACCATACCTA GTATAAACAA TGAGACACCA 421GGGATTAGAT ATCAATATAA TGTGCTTCCA CAGGGATGGA AAGGATCACC ATCAATATTC 481CAAAGTAGTA TGACAAAAAT CTTAGAGCCC TTTAGGGCAC AAAATCCAGA ATTGGTTATT 541TATCAATATA TGGATGACTT GTATGTAGGA TCCGACTTAG AAATAGGGCA GCATAGAGCA 601AAAATAGAGG AGTTAAGAAA ACATCTATTG AGGTGGGGAT TTACCACACC AGACAAGAAA 661CATCAGAAAG AACCTCCATT TCTTTGGATG GGGTATGAAC TCCATCCTGA CAAATGGACA 721GTACAGCCTA TAAAGCTGCC AGAAAAGGAT AGCTGGACTG TTAATGATAT ACAGAAGTTA 781GTGGGAAAAC TAAACTGGGC AAGTCAGATT TACAAAGGGA TTAAAGTAAG GCAGCTGTGT 841AGACTCCTTA GGGGAGCCAA AGCACTAACA GACATAGTAC CACTGACTGA AGAAGCAGAA 901TTAGAATTGG CAGAGAACAG GGAAATTCTA AAAGAACCAG TACATGGAGT ATATTATGAC 961TCA

Genome Location: 2512 . . . 3477

Additional Similar Nucleotide Examples: Accession Numbers: AY510056,AY510047, AY589935, AF468458

Protein: Accession Number: AAR92448

HIV-1 Subtype D Genome

Accession Number: AY322189, AY773341, AJ320484

HIV-1 Protease

Exemplary Sequence (SEQ ID NO: 94) 1CCTCAAATCA CTCTTTGGCA ACGACCCCTT GTCACAGTAA RGATAGGGGG ACAACTAAAG 61GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTGG AAGAAATGAA TTTGCCAGGA 121AAATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGCTTTA TCAAAGTAAG ACAGTATGAT 181CAAATACTTG TAGAAATCTG TGGATATAAG GCTATAGGTA CAGTGTTAGT AGGACCTACA 241CCTGTCAACA TAATTGGAAG AAATTTGTTG ACTCAGATTG GTTGCACTTT AAATTTT

Genome Location: 1719 . . . 2015

Additional Similar Nucleotide Examples: Accession Numbers: AJ296664

Protein: Accession Number: CAC03695

HIV-1 Reverse Transcriptase

Exemplary Sequence (SEQ ID NO: 95) 1CCAATTAGTC CTATTGAAAC TGTACCAGTA AAATTAAAGC CAGGGATGGA TGGCCCAAAA 61GTTAAACAAT GGCCGTTAAC AGAAGAAAAA ATAAAAGCAC TAACAGAAAT TTGTACAGAA 121ATGGAAAAGG AAGGAAAAAT TTCAAGAATT GGGCCTGAAA ATCCATACAA TACTCCAATA 181TTTGCCATAA AGAAAAAAGA CAGTACTAAR TGGAGAAAAT TAGTAGATTT TAGAGAACTT 241AATAAGAGAA CTCAAGACTT CTGGGAAGTT CAACTAGGAA TACCACATCC TGCAGGGCTA 301AAAAAGAAAA AATCAGTAAC AGTACTGGAT GTGGGWGATG CATATTTTTC AGTTCCCTTA 361TATGAAGACT TTAGAAAATA TACTGCATTC ACCATACCYA GTATAAATAA TGAGACACCA 421GGAATTAGAT ATCAGTACAA TGTGCTTCCA CAAGGATGGA AAGGATCACC GGCAATATTT 481CAAAGTAGCA TGACAAAAAT CTTAGAACCT TTTAGAAAAC AAAATCCAGA AATGGTGATC 541TATCAATACA TGGATGATTT GTATGTAGGA TCTGACTTAG AAATAGGGCA GCATAGAATA 601AAAATAGAGG AATTAAGGGA ACACTTATTG AAGTGGGGAT TTACCACACC AGACAAAAAG 661CATCAGAAAG AACCCCCATT TCTTTGGATG GGTTATGAAC TCCATCCGGA TAAATGGACA 721GTACAGCCTA TAAAACTGCC AGAAAAAGAA AGCTGGACTG TCAATGATAT ACAGAAGTTA 781GTGGGAAAAT TAAATTGGGC AAGTCAGATT TATCCAGGAA TTAAAGTAAG ACAATTATGC 841AAATGCATTA GGGGAGCCAA AGCACTGACA GAAGTAGTAC CACTGACAGAAGAAGCAGAA 901TTAGAACTGG CAGAAAACAG AGAAATTCTA AAAGAACCAG TACATGGAGT GTATTATGAT 961CCA

Genome Location: 2016 . . . 2978

Additional Similar Nucleotide Examples: Accession Numbers: AF388101

Protein: Accession Number: AAL84043

Methods

Provided are methods for the specific detection of several mutations inHIV. Mutations in both the reverse transcriptase and the protease of HIVcan be detected using the methods described herein. The methods arehighly sensitive and specific. Specific examples of such methods aredescribed. However, it is recognized that modifications of theexemplified methods using the alternative methods disclosed can beroutinely accomplished. Any viral RNA can be used in the presentinvention. Such RNA is not limited to that obtained from plasma orserum, but can also be intracellular RNA that has not been packaged.Detection can be achieved so long as any of the disclosed forwardprimers are paired with any of the reverse primers for a given mutation.The following methods describe specific sets of primers that achieveespecially sensitive levels of detection.

A method for detecting the 103N mutation in the reverse transcriptase ofHIV-1 is provided, comprising (a) reverse transcribing RNA extractedfrom HIV-1 with a primer selected from the group consisting of SEQ IDNO:3 and SEQ ID NO:6 to produce a reverse transcription reactionproduct; (b) contacting the reverse transcription product of step (a)with a primer set selected from the group consisting of SEQ ID NOS:1,2,4and 5 to produce a DNA product; and (c) contacting the DNA product ofstep (b) with a reverse primer and a primer set selected from the groupconsisting of SEQ ID NOS:22,23,24 and 25 and SEQ ID NOS:59,60 and 61 toamplify HIV-1 DNA containing the 103N mutation. The reverse primer isroutinely selected based on the well-known criteria for such selections,which are described herein and elsewhere. For example, the reverseprimer can be a primer comprising or consisting of SEQ ID NO:26. In themethods disclosed, the presence of an amplification signal within acertain number of cycles after signal detection in the total copy PCRreaction indicates the presence of the respective mutation. This method,for use with an RNA template, detects the 103N mutation in either orboth of Subtype B and Subtype C. SEQ ID NOS: 4 and 5 are forward RT-PCR(for RNA) and primary PCR (for DNA) primers for Subtype C. SEQ ID NO:4includes protease sequences while SEQ ID NO:5 is for reversetranscriptase only. SEQ ID NOS: 1 and 2 are forward RT-PCR (for RNA) andprimary PCR (for DNA) primers for Subtype B. SEQ ID NO:1 includesprotease sequences while SEQ ID NO:2 is for reverse transcriptase only.

Details of the RT-PCR (steps (a) and (b)) and secondary PCR (step (c))for the detection methods starting with RNA are described in theExamples. In step (c) of these methods, a set of primers is used,including at least a primer pair comprising a reverse primer and one ofthe disclosed forward primers for the respective mutation. In step (b)of the methods starting with RNA, the choice of amplifying both thereverse transcriptase and the protease are provided by an exemplaryprimary PCR forward primer that includes protease and an exemplaryprimary forward primer for reverse transcriptase only.

Each forward primer disclosed for the RT-PCR reaction or the primary PCRreaction in the methods disclosed works independently. If a proteaseanalysis is to be done, then the F 1 primers must be used for the RT-PCRor primary PCR steps. Reverse transcriptase analyses can be performedfrom the F2+reverse primer products alone (the F2 primers are slightlymore sensitive than the F1 primers, thus can provide the user with amore sensitive test). In step (b) of the methods starting with RNA,there is reverse primer remaining in the reaction product from step (a).

The RT step of the present methods can utilize RT primers other thanthose described. The only requirement is that the primers generate atemplate in the relevant region of the reverse transcriptase gene or inthe protease gene or both.

A further method for detecting the 103N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1,2,4 and 5 to amplify the DNA; and (b) contacting the amplified DNAof step (a) with a reverse primer and a primer set selected from thegroup consisting of SEQ ID NOS:22,23,24 and 25 and SEQ ID NOS:59,60 and61 to amplify HIV-1 DNA containing the 103N mutation. The reverse primeris routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:26.This method, for use with a DNA template, detects the 103N mutation ineither or both of Subtype B and Subtype C.

Details of the primary PCR and secondary PCR steps for the detectionmethods starting with DNA are described in the Examples. In step (b) ofthese methods, a set of primers is used, including at least a primerpair comprising a reverse primer and one of the disclosed forwardprimers for the respective mutation. In step (a) of the methods startingwith DNA, the choice of amplifying both the reverse transcriptase andthe protease are provided by an exemplary primary PCR forward primerthat includes protease and an exemplary forward primer for reversetranscriptase only. Each forward primer disclosed for the primary PCRreaction in the method beginning with DNA works independently. Thus, theRT-only primer and the protease-included primer can be usedindependently with a reverse primer. If a protease analysis is to bedone, then the F1 primers must be used for the RT-PCR or primary PCRsteps. Reverse transcriptase analyses can be performed from theF2+reverse primer products alone (the F2 primers are slightly moresensitive than the F1 primers, thus can provide the user with a moresensitive test).

Amplification methods are provided that include a probe for use in areal time PCR reaction. The methods can thus include the use of aforward primer, a reverse primer and a probe. For example, anamplification method is provided comprising a forward primer or amixture of forward primers that amplifies the protease 90M, and thereverse transcriptase 103N, 65R, and 70R mutations, wherein the methodfurther comprises using an oligonucleotide having the nucleotides as setforth in SEQ ID NO:9. This is an example of a probe that can be used inany of these mutation-specific PCR reactions. This probe can also beused in the total copy PCR reaction.

A method for detecting a Subtype B 184V mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:33,34 and 35 and a reverse primer to amplify HIV-1 DNA containinga Subtype B 184V mutation. The reverse primer is routinely selectedbased on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:36.

A method for detecting a Subtype B 184V mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:33,34 and 35 and areverse primer to amplify HIV-1 DNA containing a Subtype B 184Vmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:36.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 181C and Subtype B184V mutations, wherein the method further comprises using anoligonucleotide having the nucleotides as set forth in SEQ ID NO:32.This is an example of a probe that can be used in either of thesemutation-specific PCR reactions.

A method for detecting a Subtype B 41L mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:62,63,64 and 65 and a reverse primer to amplify HIV-1 DNAcontaining a Subtype B 41L mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:66.

A method for detecting a Subtype B 41L mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:63,96,97,64, and 65 and a reverse primer to amplify HIV-1 DNAcontaining a Subtype B 41L mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ 1D NO:66.

A method for detecting a Subtype B 41L mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS: 62,63,64 and 65 and areverse primer to amplify HIV-1 DNA containing a Subtype B 41L mutation.The reverse primer is routinely selected based on the well-knowncriteria for such selections, which are described herein and elsewhere.For example, the reverse primer can be a primer comprising or consistingof SEQ ID NO:66.

A method for detecting a Subtype B 41L mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS: 63,96,97,64, and 65and a reverse primer to amplify HIV-1 DNA containing a Subtype B 41Lmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:66.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 41L mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NO:67. This is an example of a probethat can be used in mutation-specific PCR reactions for this mutation.

A method for detecting a Subtype B 65R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer comprising SEQ IDNO:10 and a reverse primer to amplify HIV-1 DNA containing a Subtype B65R mutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:11.

A method for detecting a Subtype B 65R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer comprising SEQ IDNO:98 and a reverse primer to amplify HIV-1 DNA containing a Subtype B65R mutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:11.

A method for detecting a Subtype B 65R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer comprising SEQ ID NO:10 and a reverse primer toamplify HIV-1 DNA containing a Subtype B 65R mutation. The reverseprimer is routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:11.

A method for detecting a Subtype B 65R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer comprising SEQ ID NO:98 and a reverse primer toamplify HIV-1 DNA containing a Subtype B 65R mutation. The reverseprimer is routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:11.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 65R mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NOS:9, 68, or 99. These are examplesof probes that can be used in mutation-specific PCR reactions for thismutation.

A method for detecting a Subtype B 67N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:69 and 70 and a reverse primer to amplify HIV-1 DNA containing aSubtype B 67N mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:8.

A method for detecting a Subtype B 67N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:69 and 70 and a reverseprimer to amplify HIV-1 DNA containing a Subtype B 67N mutation. Thereverse primer is routinely selected based on the well-known criteriafor such selections, which are described herein and elsewhere. Forexample, the reverse primer can be a primer comprising or consisting ofSEQ ID NO:8.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 67N mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NO:68. This is an example of a probethat can be used in mutation-specific PCR reactions for this mutation.

A method for detecting a Subtype B 69T mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:12 and 13 and a reverse primer to amplify HIV-1 DNA containing aSubtype B 69T mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:14.

A method for detecting a Subtype B 69T mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:12 and 71 and a reverse primer to amplify HIV-1 DNA containing aSubtype B 69T mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:8.

A method for detecting a Subtype B 69T mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:12 and 13 and a reverseprimer to amplify HIV-1 DNA containing a Subtype B 69T mutation. Thereverse primer is routinely selected based on the well-known criteriafor such selections, which are described herein and elsewhere. Forexample, the reverse primer can be a primer comprising or consisting ofSEQ ID NO:14.

A method for detecting a Subtype B 69T mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:12 and 71 and a reverseprimer to amplify HIV-1 DNA containing a Subtype B 69T mutation. Thereverse primer is routinely selected based on the well-known criteriafor such selections, which are described herein and elsewhere. Forexample, the reverse primer can be a primer comprising or consisting ofSEQ ID NO:8.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 69T mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NOS:9 or 68. These are examplesprobes that can be used in mutation-specific PCR reactions for thismutation.

A method for detecting a Subtype B 70R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:16,17,18 and 19 and a reverse primer to amplify HIV-1 DNAcontaining a Subtype B 70R mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:20.

A method for detecting a Subtype B 70R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NO:2 and a reverse primer to amplify HIV-1 DNA containing a Subtype B70R mutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NOS:72 and 73.

A method for detecting a Subtype B 70R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NO:100 and a reverse primer to amplify HIV-1 DNA containing a SubtypeB 70R mutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NOS:72 and 73.

A method for detecting a Subtype B 70R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:16,17,18 and 19 and areverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.The reverse primer is routinely selected based on the well-knowncriteria for such selections, which are described herein and elsewhere.For example, the reverse primer can be a primer comprising or consistingof SEQ ID NO:20.

A method for detecting a Subtype B 70R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NO:2 and a reverse primerto amplify HIV-1 DNA containing a Subtype B 70R mutation. The reverseprimer is routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NOS:72and 73.

A method for detecting a Subtype B 70R mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NO:100 and a reverse primerto amplify HIV-1 DNA containing a Subtype B 70R mutation. The reverseprimer is routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NOS:72and 73.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 70R mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NO:9 or 67. This is an example of aprobe that can be used in mutation-specific PCR reactions for thismutation.

A method for detecting a Subtype B 103N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:22,23,24 and 25 and a reverse primer to amplify HIV-1 DNAcontaining a Subtype B 103N mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ 1D NO:26.

A method for detecting a Subtype B 103N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:22,23,24 and 25 and areverse primer to amplify HIV-1 DNA containing a Subtype B 103Nmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:26.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 103N mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NO:9. This is an example of a probethat can be used in mutation-specific PCR reactions for this mutation.

A method for detecting a Subtype B 181C mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:28 and 29 and a reverse primer to amplify HIV-1 DNA containing aSubtype B 181C mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:30.

A method for detecting a Subtype B 181C mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS: 28 and 29 and areverse primer to amplify HIV-1 DNA containing a Subtype B 181Cmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:30.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype B 181C mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NO:32. This is an example of a probethat can be used in mutation-specific PCR reactions for this mutation.

A method for detecting a Subtype B 215T mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 or SEQ ID NOS:74 and 75 or SEQ IDNOS:101 and 75 to produce a DNA product; (c) contacting the DNA productof step (b) with a reverse primer and a primer selected from the groupconsisting of SEQ ID NOS:38 and 39 to amplify HIV-1 DNA containing aSubtype B 215 mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:45.

A method for detecting a Subtype B 215 mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:1 and 2 to produce a DNA product; (c)contacting the DNA product of step (b) with a reverse primer and aprimer selected from the group consisting of SEQ ID NOS:40,41,42,43 and44 to amplify HIV-1 DNA containing a Subtype B 215 mutation. The reverseprimer is routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NO:45.

A method for detecting a Subtype B 215 mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a reverse primer and a primer selected from the groupconsisting of SEQ ID NOS:40,41,42,43 and 44 to amplify HIV-1 DNAcontaining a Subtype B 215 mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:45.

In the present methods of detecting a mutation at position Subtype B215, any or all of the Y, F, S, C or D mutations can be detected. Thus,to detect any mutation at this position, the forward primers can be usedtogether in the reaction mixture. To detect a specific mutation, theforward primer for that mutation would be used alone. Specificcombinations of mutations at 215 can be identified by using the desiredsubset of the disclosed forward primers.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies Subtype B 215 mutations,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NOS:47, 76, or 77. This is an exampleof a probe that can be used in mutation-specific PCR reactions for thismutation.

A method for detecting the 30N mutation in the protease of HIV-1 SubtypeB is provided, comprising (a) reverse transcribing RNA extracted fromHIV-1 with a primer comprising SEQ ID NO:3 to produce a reversetranscription reaction product; (b) contacting the reverse transcriptionproduct of step (a) with a primer selected from the group consisting ofSEQ ID NOS:1 and 2 to produce a DNA product; and (c) contacting the DNAproduct of step (b) with a primer set comprising SEQ ID NOS:48 and 49and a reverse primer to amplify HIV-1 DNA containing the 30N mutation.The reverse primer is routinely selected based on the well-knowncriteria for such selections, which are described herein and elsewhere.For example, the reverse primer can be a primer comprising or consistingof SEQ ID NO:50.

A method for detecting the 30N mutation in the protease of HIV-1 SubtypeB is provided, comprising (a) contacting DNA with a reverse primer and aprimer selected from the group consisting of SEQ ID NOS:1 and 2 toamplify the DNA; and (b) contacting the amplified DNA of step (a) with aprimer set comprising SEQ ID NOS:48 and 49 and a reverse primer toamplify HIV-1 DNA containing the 30N mutation. The reverse primer isroutinely selected based on the well-known criteria for such selections,which are described herein and elsewhere. For example, the reverseprimer can be a primer comprising or consisting of SEQ ID NO:50.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies the protease 30N mutation ofHIV-1 Subtype B, wherein the method further comprises the use of anoligonucleotide having the nucleotides as set forth in SEQ ID NO:52.This is an example of a probe that can be used in imitation-specific PCRreactions for this mutation.

A method for detecting the 90M mutation in the protease of HIV-1 SubtypeB is provided, comprising (a) reverse transcribing RNA extracted fromHIV-1 with a primer comprising SEQ ID NO:3 to produce a reversetranscription reaction product; (b) contacting the reverse transcriptionproduct of step (a) with a primer selected from the group consisting ofSEQ ID NOS:1 and 2 to produce a DNA product; and (c) contacting the DNAproduct of step (b) with a primer set comprising SEQ ID NOS:53,54, and55 and a reverse primer to amplify HIV-1 DNA containing the 90Mmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:56.

A method for detecting the 90M mutation in the protease of HIV-1 SubtypeB is provided, comprising (a) reverse transcribing RNA extracted fromHIV-1 with a primer comprising SEQ ID NO:3 to produce a reversetranscription reaction product; (b) contacting the reverse transcriptionproduct of step (a) with a primer selected from the group consisting ofSEQ ID NOS:1 and 2 to produce a DNA product; and (c) contacting the DNAproduct of step (b) with a primer set comprising SEQ ID NOS: 55,78,79,and 80 and a reverse primer to amplify HIV-1 DNA containing the 90Mmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:81.

A method for detecting the 90M mutation in the protease of HIV-1 SubtypeB is provided, comprising (a) contacting DNA with a reverse primer and aprimer selected from the group consisting of SEQ ID NOS:1 and 2 toamplify the DNA; and (b) contacting the amplified DNA of step (a) with aprimer set comprising SEQ ID NOS:53,54, and 55 and a reverse primer toamplify HIV-1 DNA containing the 90M mutation. The reverse primer isroutinely selected based on the well-known criteria for such selections,which are described herein and elsewhere. For example, the reverseprimer can be a primer comprising or consisting of SEQ ID NO:56.

A method for detecting the 90M mutation in the protease of HIV-1 SubtypeB is provided, comprising (a) contacting DNA with a reverse primer and aprimer selected from the group consisting of SEQ ID NOS:1 and 2 toamplify the DNA; and (b) contacting the amplified DNA of step (a) with aprimer set comprising SEQ ID NOS:55,78,79, and 80 and a reverse primerto amplify HIV-1 DNA containing the 90M mutation. The reverse primer isroutinely selected based on the well-known criteria for such selections,which are described herein and elsewhere. For example, the reverseprimer can be a primer comprising or consisting of SEQ ID NO:81.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies the protease 90M mutation,wherein the method further comprises the use of an oligonucleotidehaving the nucleotides as set forth in SEQ ID NOS:58 or 82. These areexamples probes that can be used in mutation-specific PCR reactions forthis mutation.

A method for detecting a Subtype C 103N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:59,60, and 61 and a reverse primer to amplify HIV-1 DNAcontaining a Subtype C 103N mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:26.

A method for detecting a Subtype C 103N mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS: 59,60, and 61 and areverse primer to amplify HIV-1 DNA containing a Subtype C 103Nmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:26.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype C 103N mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NO:9. This is an example of a probethat can be used in mutation-specific PCR reactions for this mutation.

A method for detecting a Subtype C 181C mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:83 and 84 and a reverse primer to amplify HIV-1 DNA containing aSubtype C 181C mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:85.

A method for detecting a Subtype C 181C mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:3 and 4 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS:83 and 84 and a reverseprimer to amplify HIV-1 DNA containing a Subtype C 181C mutation. Thereverse primer is routinely selected based on the well-known criteriafor such selections, which are described herein and elsewhere. Forexample, the reverse primer can be a primer comprising or consisting ofSEQ ID NO:85.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype C 103N mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NOS:86 or 87. These are examples ofprobes that can be used in mutation-specific PCR reactions for thismutation.

A method for detecting a Subtype C 184V mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:88 and 89 and a reverse primer to amplify HIV-1 DNA containing aSubtype C 184V mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NO:85.

A method for detecting a Subtype C 184V mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) reverse transcribingRNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to producea reverse transcription reaction product; (b) contacting the reversetranscription product of step (a) with a primer selected from the groupconsisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c)contacting the DNA product of step (b) with a primer set comprising SEQID NOS:102,103 and 104 and a reverse primer to amplify HIV-1 DNAcontaining a Subtype C 184V mutation. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:85.

A method for detecting a Subtype C 184V mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS: 88 and 89 and areverse primer to amplify HIV-1 DNA containing a Subtype C 184Vmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:85.

A method for detecting a Subtype C 184V mutation in the reversetranscriptase of HIV-1 is provided, comprising (a) contacting DNA with areverse primer and a primer selected from the group consisting of SEQ IDNOS:1 and 2 to amplify the DNA; and (b) contacting the amplified DNA ofstep (a) with a primer set comprising SEQ ID NOS: 102,103 and 104 and areverse primer to amplify HIV-1 DNA containing a Subtype C 184Vmutation. The reverse primer is routinely selected based on thewell-known criteria for such selections, which are described herein andelsewhere. For example, the reverse primer can be a primer comprising orconsisting of SEQ ID NO:85.

An amplification method is provided comprising a forward primer ormixture of forward primers that amplifies a Subtype C 184V mutation,wherein the method further comprises using an oligonucleotide having thenucleotides as set forth in SEQ ID NOS:86 or 87. These are examples ofprobes that can be used in mutation-specific PCR reactions for thismutation.

A method for amplifying the reverse transcriptase of HIV-1 is provided,comprising (a) reverse transcribing RNA extracted from HIV-1 with aprimer comprising SEQ ID NO:3 to produce a reverse transcriptionreaction product; (b) contacting the reverse transcription product ofstep (a) with a primer selected from the group consisting of SEQ IDNOS:1 and 2 to produce a DNA product; and (c) contacting the DNA productof step (b) with a primer comprising SEQ ID NO:7 and a reverse primer toamplify a region encoding the reverse transcriptase of HIV-1. Thereverse primer is routinely selected based on the well-known criteriafor such selections, which are described herein and elsewhere. Forexample, the reverse primer can be a primer comprising or consisting ofSEQ ID NO:8. This can be a common amplification method of the invention.The total copy reaction can be used to provide the baseline for themutation-specific real time PCR reactions disclosed herein.Alternatively, matched wildtype primers can be used as a control, as isknown to one skilled in the art.

A method for amplifying the reverse transcriptase of HIV-1 is provided,comprising (a) contacting DNA with a reverse primer and a primerselected from the group consisting of SEQ ID NOS:1 and 2 to amplify theDNA; and (b) contacting the amplified DNA of step (a) with a primercomprising SEQ ID NO:7 and a reverse primer to amplify a region encodingthe reverse transcriptase of HIV-1. The reverse primer is routinelyselected based on the well-known criteria for such selections, which aredescribed herein and elsewhere. For example, the reverse primer can be aprimer comprising or consisting of SEQ ID NO:8.

The amplification methods disclosed herein can utilize reverse primersother than those exemplified. The exemplified reverse primers were foundto work well. However, the requirements of the reverse primer in thepresent method are typical of reverse primers designed and usedroutinely, and other reverse primers can be routinely made and used. Itis expected that the reverse primer will be within about 40 to 250 basesfrom the forward primer. It is also expected that the reverse primerwill be positioned in a stable location lacking variability to a degreethat would impede binding. The reverse primer is most likely to belocated in the RT gene or the protease gene, but the exact location isroutinely variable based on the usual criteria for reverse primerpositioning.

Methods disclosed herein can further include detection, in the samemixture, of a specified RT mutation and a specified protease mutation.For example, provided is a method for detecting a 184V mutation in thereverse transcriptase of HIV-1 and a 90M mutation in the protease ofHIV-1, comprising (a) reverse transcribing RNA extracted from HIV-1 witha primer comprising SEQ ID NO:3 to produce a reverse transcriptionreaction product; (b) contacting the reverse transcription product ofstep (a) with a primer selected from the group consisting of SEQ IDNOS:1 and 2 to produce a DNA product; and (c) contacting the DNA productof step (b) with a primer set comprising SEQ ID NOS:33,34, 35, 55,78,79, and 80 and a reverse primer to amplify HIV-1 DNA containing a184V and a 90M mutation. The reverse primer is routinely selected basedon the well-known criteria for such selections, which are describedherein and elsewhere. For example, the reverse primer can be a primercomprising or consisting of SEQ ID NOS:36 and 81.

A method for detecting a 184V mutation in the reverse transcriptase ofHIV-1 and a 90M mutation in the protease of HIV-1 is provided,comprising (a) contacting DNA with a reverse primer and a primerselected from the group consisting of SEQ ID NOS:1 and 2 to amplify theDNA; and (b) contacting the amplified DNA of step (a) with a primer setcomprising SEQ ID NOS:33,34,35,55,78,79, and 80 and a reverse primer toamplify HIV-1 DNA containing a 184V and a 90M mutation. The reverseprimer is routinely selected based on the well-known criteria for suchselections, which are described herein and elsewhere. For example, thereverse primer can be a primer comprising or consisting of SEQ ID NOS:36and 81.

A variety of technologies related to real-time (or kinetic) PCR havebeen adapted to perform point mutation and SNP detection. Mutationdetection using real-time amplification relies on the ability to detectamplified segments of nucleic acid as they are during the amplificationreaction. Three basic real-time detection methodologies exist: (i)increased fluorescence of double strand DNA specific dye binding, (ii)decreased quenching of fluorescence during amplification, and (iii)increased fluorescence energy transfer during amplification (Wittwer, C.et al. Biotechniques 22:130-138, 1997). All of these techniques arenon-gel based and each strategy is disclosed.

A variety of dyes are known to exhibit increased fluorescence inresponse to binding double stranded DNA. Production of wild type ormutation containing PCR products are continuously monitored by theincreased fluorescence of dyes such as ethidium bromide or Syber Greenas they bind to the accumulating PCR product. Note that dye binding isnot selective for the sequence of the PCR product, and high non-specificbackground can give rise to false signals with this technique.

A second detection technology for real-time PCR, known generally asexonuclease primers (e.g., TaqMan® probes), utilizes the 5′ exonucleaseactivity of thermostable polymerases such as Taq to cleave dual-labeledprobes present in the amplification reaction (Wittwer, C. et al.Biotechniques 22:130-138, 1997; Holland, P et al PNAS 88:7276-7280,1991, incorporated herein by reference). While complementary to the PCRproduct, the probes used in this assay are distinct from the PCR primerand are dually-labeled with both a molecule capable of fluorescence anda molecule capable of quenching fluorescence. When the probes areintact, intramolecular quenching of the fluorescent signal within theDNA probe leads to little signal. When the fluorescent molecule isliberated by the exonuclease activity of the polymerase duringamplification, the quenching is greatly reduced leading to increasedfluorescent signal.

An additional form of real-time PCR also capitalizes on theintramolecular quenching of a fluorescent molecule by use of a tetheredquenching moiety. The molecular beacon technology utilizeshairpin-shaped molecules with an internally-quenched fluorophore whosefluorescence is restored by binding to a DNA target of interest (Kramer,R. et al. Nat. Biotechnol. 14:303-308, 1996, incorporated herein byreference). Increased binding of the molecular beacon probe to theaccumulating PCR product can be used to specifically detect SNPs presentin genomic DNA.

A final, general fluorescent detection strategy used for detection ofpoint mutations and SNP in real time utilizes synthetic DNA segmentsknown as hybridization probes in conjunction with a process known asfluorescence resonance energy transfer (FRET) (Wittwer, C. et al.Biotechniques 22:130-138, 1997; Bernard, P. et al. Am. J. Pathol.153:1055-1061, 1998, incorporated herein by reference). This techniquerelies on the independent binding of labeled DNA probes on the targetsequence. The close approximation of the two probes on the targetsequence increases resonance energy transfer from one probe to theother, leading to a unique fluorescence signal. Mismatches caused bySNPs that disrupt the binding of either of the probes can be used todetect mutant sequences present in a DNA sample.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids, which are incorporated herein in their entireties). Apreferable stringent hybridization condition for a DNA:DNA hybridizationcan be at about 68° C. (in aqueous solution) in 6×SSC or 6×SSPE followedby washing at 68° C. Stringency of hybridization and washing, ifdesired, can be reduced accordingly as the degree of complementaritydesired is decreased, and further, depending upon the G-C or A-Trichness of any area wherein variability is searched for. Likewise,stringency of hybridization and washing, if desired, can be increasedaccordingly as homology desired is increased, and further, dependingupon the G-C or A-T richness of any area wherein high homology isdesired, all as known in the art.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

This example describes the development and application of real-timePCR-based point mutation assays for the 103N and 184V mutations in thereverse transcriptase (RT) of HIV-1. The assay measures the differentialamplifications of total copy and mutation-specific reactions that targetcodons of interest. In evaluating mutation-containing plasmids dilutedin backgrounds of wild type plasmid, the assays were able to achieve amutation detection limit of 0.04% and 0.2% 103N and 184V, respectively.To evaluate the performance of these assays with clinical specimens, 77known wild-type samples were first analyzed. None of the wild-typesamples was positive for the 184V mutation, while one sample (1.3%)scored positive for 103N. Conversely, in plasma samples known to haveviruses carrying the 103N mutation and/or the 184V mutation, 103N wasdetected in 54 of 55 positive specimens (98%), and 184V in 65 of 67(97%). To determine whether any mutation-containing samples wereundetected by conventional sequence analysis, the present assays wereapplied to a test population of HIV-1-positive treatment-naïve personsdocumented to have RT mutations other than 103N and 184V. The 103N assaydetected 4 positive samples (2.4%) in 165 plasma samples previouslyfound absent of 103N (clones are currently being screened for thepresence of low-level mutants). Likewise, in 173 samples previouslydetermined to be negative for the 184V mutation, three samples scoredpositive (1.1%) for 184V by this assay. Two were later verified to havethe mutation (at frequencies of 1.4% and 5.5%) by sequence analysis ofclones. The data demonstrate that currently used sequence analysis isfailing to detect resistant HIV-1 present as minority species inclinical specimens. The data also demonstrate that these real-time PCRassays for the detection of the 103N and 184V mutations are sensitiveand specific. Given the low cost, high-throughput capability, andgreater sensitivity than conventional testing, these assays will beuseful for detecting drug resistance-associated mutations and could aidin the clinical management of HIV-1 infection.

Clinical Samples

Wild type HIV-1 subtype B samples were obtained from the plasma of 23treatment-naïve persons (2) with no detectable resistance mutations, andfrom 54 sera collected in the early 1980's, prior to the development ofantiretroviral drugs. 67 specimens confirmed by sequence analysis tohave virus carrying the mutation comprised mutation-positive samples.The test population encompassed a second group of 173 treatment-naïvepatients (partially referenced in 2), all with RT mutations other than103N or 184V. Approximately 17% of the treatment-naïve specimens werefrom persons documented to be recently infected. Results obtained fromevaluation of the wild type and mutation-confirmed samples were used todefine the assay cutoffs.

Reverse Transcriptase-PCR

HIV-1 genomic RNA was extracted (Qiagen UltraSens RNA kit) from patientplasma or serum. Primary amplifications of HIV-1 template were generatedby reverse transcriptase-PCR using primers that demarcated the firsthalf of the RT sequence, or when desired, the forward primer was shiftedupstream to also include the entire protease region. The minimum copynumbers from which these reactions could successfully amplify were 5 and10 RNA copies, respectively.

Real-Time PCR

Baseline measurements for viral copies in test samples were determinedusing HIV-1 RT total copy primers with a total copy probe (FIG. 1A).Preliminary analysis for detecting the presence of the mutation wasperformed using primer mixtures to compensate for polymorphicvariability in the primer binding sites. The 103N-specific mixtureincorporated four different primers, and the 184V-specific reaction usedthree primers. The primers can be mixed at optimal ratios to equilibratethe differences in primer affinities. Examples of such ratios areprovided below. It is recognized that the optimization of primer ratiosis routine given the teaching of the primers and primer mixturesthemselves. One of skill can envision alternative ratios.

The mutation-specific primers were designed to maximize specificity forannealing to the mutated nucleotide(s), thus having a reduced affinityfor wild type sequences (3,4). The probes for each reaction were 5′labeled with FAM and quenched with QSY-7. The choice of fluorophore andquencher can be routinely varied. Common fluorophores include HEX, ROX,Texas Red, TAMRA, JOE, Cy3, Cy5, SYBR and VIC. There are others thatoften overlap the above spectra and can be used. The Bio-Rad fluorophoretable contains a more complete listing of fluorophores that can be usedfor this method.

Degradation of the fluorescent probes during chain elongation removesthe fluorophore from the proximity of the quencher and generates thefluorescent signals, reported as relative fluorescent units (RFU), thatincrease with each amplification cycle (FIG. 1B). The cycle number wherethe fluorescence emission exceeds the software-derived threshold iscalled the threshold cycle (CT) and is the unit of measure whencomparing the differences in amplification levels (ΔCT) of the totalcopy and mutation-specific reactions.

All reactions were performed using an iCycler real-time PCR system(Bio-Rad) and AmpliTaq Gold polymerase (Applied Biosystems). Anyhot-start polymerase will work in this method. The differences betweenthese are in their ability to extend from mismatched primers. Assaycutoffs (limits) are established for each polymerase. Other usablepolymerases include, but are not limited to, AmpliTaq Platinum (AppliedBiosystems) and iTaq (Bio-Rad). PCR annealing was at 50° C. for 15seconds and extension at 60° C. for 30 seconds (See detailed PCTprotocol below). Samples that were just above the cutoff (<2 CT) wereagain analyzed using individual primers for the mutation in order toincrease the sensitivity of the test.

Assay Sensitivity

Assay detection limits were tested against dilutions ofmutation-containing plasmid clones and from PCR products from bothlab-adapted HIV-1 and patient-derived mutant virus spiked into abackground of wild type virus template. The amounts of mutant inputcomprised 100%-0.001% of the total virus population.

Protocol for HIV Real-Time PCR Point Mutation Assays

I. Sample Preparation

-   -   Extract viral RNA from 1000: I plasma or proviral DNA from        ˜7.5×10⁵ cells (Qiagen Ultrasensitive ViralAmp or Promega Wizard        Genomic DNA kits, respectively)

II. For RNA Template

-   -   Primary (general) RT-PCR—    -   Use 5:1 extracted RNA per RT-PCR as follows:    -   (RT step)—        -   Per reaction, add 5:1 RNA to a total of 40:1 of reagents            prepared as follows:

DEPC water 11:1 10x buffer II  4:1 MgCl₂  8:1 (final conc. 5 mM) dNTPs 6:1 (final conc. 1.5 mM each) Reverse primer^(a)  4:1 (final conc. 400nM) RNase inhibitor*  1:1 (20 U final) MuLV RT*  1:1 (50 U final) +5:1sample RNA 40:1 total *Heat the RNA in aliquotted mastermix for 2minutes at 94° C. then immediately place on ice prior to adding theRNase inhibitor and RT.

RT reaction: 39° C. for 1 hour, 94° C. for 5 minutes, 4° C. hold

-   -   (PCR step)—        -   Add the entire RT reaction to 60:1 PCR mix prepared per            reaction as follows:

sterile water  48:1 10x buffer  8:1 Forward primer^(a)  3:1 (final conc.120 nM) AmpliTaq LD  1:1 (5 U final)  60:1 mix +40:1 RT reaction 100:1total PCR

-   -   PCR controls (in duplicate): 1) water=blank,        -   2) uninfected human DNA=(−),        -   3) plasmid @ 1000 copies/r×n spiked in human DNA=(+), or a            10⁵→10²/10:1 plasmid copy number series for quantitation            (also see IV.).^(†) ^(†)The plasmid standards can be            prepared and aliquotted as 40-cycle reactants of which 2:1            are used for the copy standards in each secondary reaction            plate.

III. For DNA Template

-   -   Primary (general) PCR—    -   Per reaction, add 10:1 DNA (a higher concentration of template        increases chances of encountering resistant proviruses) to a        total of 100:1 of reagents prepared as follows:

Sterile water  67:1 10x buffer  10:1 dNTPs  6:1 (final conc. 600: Meach) Forward primer^(a)  3:1 (final conc. 120 nM) Reverse primer^(a) 3:1 (final conc. 120 nM) AmpliTaq (LD)  1:1 (5 U final) +10:1 sampleDNA 100:1 total PCR

-   -   The 100:1 primary PCR reaction may be diluted (1:10-1:20) prior        to the real-time PCR reaction to ensure the secondary reaction        is not overloaded with template and to provide sufficient        template for future studies.

1° PCR conditions: 95° C. for 4 minutes  

95° C. for 45 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes × 40cycles  

  72° C. for 5 minutes  

4° C. hold

IV. Mutation-Specific (Secondary) Real-Time PCR

-   -   Principle—A sequence-specific probe, labeled with a fluorophore        and a quencher, anneals to a region flanked by locus-specific        primers. PCR extension from the primers degrades the intervening        probe releasing the quencher from the proximity of the        fluorophore, thus increasing the level of detectable        fluorescence. The amplification cycle at which the level of        product (i.e., amount of degraded probe) is measurable above        background, is the threshold cycle (TC). This value directly        correlates with the amount of starting template and is the unit        of measure when making comparisons between amplification levels.

Procedure—

-   -   Use 2:1 of each primary reaction in duplicate reactions of both        a total copy and mutation-specific hot-start real-time PCR.        Prepare for each of the secondary reactions:

sterile water 30.5:1 10x buffer   5:1 Forward primer(s)^(b,c,d)   4:1(final conc. 320 nM) Reverse primer   4:1 (final conc. 320 nM) dNTPs  2:1 (final conc. 400: M) fluoro-probe   2:1 (final conc. 160 nM)AmpliTaq Gold  0.5:1 (2.5 U final) (Perkin-Elmer/ABI)   +2:1 primaryreaction   50:1 total PCR reaction

2° Real-time PCR conditions: 95° C. for 11 minutes (includesnormalization time)  

95° C. for 30 seconds, 50° C. for 15 seconds, 59° C. for 30 seconds × 50cycles  

4° C. hold

-   -   The mutation-specific tests (c) can be qualitative, by comparing        to the common (total copy) primers (b) using only the 1000 copy        positive control, or quantitative, by using the wild type and        mutation-inclusive plasmid copy number dilution series. The        quantitation can be performed without or in conjunction with a        separate mutation-independent (mi) test (d), for quantitation by        comparing the CT of the mutation reaction to the CT of the        primer complementary to the shared overlapping sequence (i.e.,        same locus) (for examples of mi primers, see the primer list        below).    -   All mutation-specific PCRs are evaluated relative to the        concomitant total copy (or wild type) reaction, the difference        being ΔCT. Mutation-specific reactions with a ΔCT below the        experimentally derived cutoff are scored positive.

An advantage of the present invention is in detection sensitivity of thevarious subtypes of HIV from various countries of the world. Forexample, the sets disclosed in the primer set below are particularlysensitive to detection of HIV subtypes across the spectrum of HIV.

ReTi-HIV Assay Oligonucleotide List:

^(a) Primers for the RT-PCR reaction: Subtype BRTPF1 (includes protease) (SEQ ID NO: 1)5′-CCT CAG ATC ACT CTT TGG CAA CG RTPF2 (only RT) (SEQ ID NO: 2)5′-AAA GTT AAA CAA TGG CCA TTG ACA G RTPREV (SEQ ID NO: 3)5′-ATC CCT GCA TAA ATC TGA CTT GC Subtype C RTPF1C (includes protease)(SEQ ID NO: 4) 5′-CCT CAA ATC ACT CTT TGG CAG CG RTPF2C (RT only)(SEQ ID NO: 5) 5′-AGG TTA AAC AAT GGC CAT TGA CAG AAG  RTP-RC(SEQ ID NO: 6) 5′-CTG GGT AAA TCT GAC TTG CCC A

Primers below are for the listed mutations. All forward primers for eachmutation can be mixed for general surveillance testing or the primerscan be used individually or mixed and matched for detecting/monitoringdistinct polymorphisms associated with that mutation. The primerproportions exemplified for these mixtures are routinely adjustableusing the optimization methods routinely practiced in this field.

Codons Label Oligonucleotide sequence^(§) ^(b) Common^(@) ComFWD5′-CTT CTG GGA AGT TCA ATT AGG AAT ACC (SEQ ID NO: 7) (copy number)ComREV 5′-CCT GGT GTC TCA TTG TTT ATA CTA GGT (SEQ ID NO: 8) probe^(#)5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TYT CAR TTC CCT TA (SEQ ID NO: 9)^(c)Mutation Subtype B Reverse transcriptase 41L Set 1 41L F15′-AAA AGC ATT ART RGA AAT YTG TRC AGG AC (SEQ ID NO: 62) 41L F25′-AAT AAA AGC ATT ART RGA AAT YTG TRC AGC AT (SEQ ID NO: 63) 41L F35′-TAA AAG CAT TAR TRG AAA TYT GTR CAK GTC (SEQ ID NO: 64) 41L F45′-AAG CAT TAR TRG AAA TYT GTR CAK GGC (SEQ ID NO: 65) 41L REV5′-CCT AAT TGA ACT TCC CAG AAG TC (SEQ ID NO: 66) 41L probe5′-FAM-TTG GGC CTG AAA A“T”C CAT ACA ATA CTC CAG TAT TT (SEQ ID NO: 67)41L Set 2 41L F25′-AAT AAA AGC ATT ART RGA AAT YTG TRC AGC AT (SEQ ID NO: 63) 41L F55′-AAT WAA AGC ATT ART RGA AAT YTG TRC WGC AT (SEQ ID NO: 96) 41L F65′-AAA AGC ATT ART RGA AAT YTG TRC AGG AC (SEQ ID NO: 97) 41L F35′-TAA AAG CAT TAR TRG AAA TYT GTR CAK GTC (SEQ ID NO: 64) 41L F45′-AAG CAT TAR TRG AAA TYT GTR CAK GGC (SEQ ID NO: 65) 41L REV5′-CCT AAT TGA ACT TCC CAG AAG TC (SEQ ID NO: 66) 41L probe5′-FAM-TTG GGC CTG AAA A“T”C CAT ACA ATA CTC CAG TAT TT (SEQ ID NO: 67)65R Set 1 65R F15′-CAT AYA ATA CYC CAR TAT TTG YCA TAA AAA G (SEQ ID NO: 10) 65R REV5′-CCT GGT GTC TCA TTG TTT ATA CTA GGT (SEQ ID NO: 11) 65R probe5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TYT CAR TTC CCT TA (SEQ ID NO: 9)65-69 probe 5′-FAM-TAG TAG ATT “T” CAG AGA ACT TAA TAA GAG AAC TCAAGA CT (SEQ ID NO: 68) 65R Set 2 65R F25′-ACA ATA CTC CAR TAT TTG CCA TAA RCA G (SEQ ID NO: 98) 65R REV5′-CCT GGT GTC TCA TTG TTT ATA CTA GGT (SEQ ID NO: 11) 65-69 probe25′-FAM- TCA GAG AAC “T” TAA TAA RAG AAC TCA AGA CTT CTGGGA (SEQ ID NO: 99) 67N 67N F25′-AAT ACT CCA RTA TTT GYC ATA ARG AAR GCA A (SEQ ID NO: 69) 67N F35′-ATA CTC CAR TAT TTG YCA TAA AGA ARG CGA (SEQ ID NO: 70) 67 REV^(#)5′-CCT GGT GTC TCA TTG TTT ATA CTA GGT (SEQ ID NO: 8) 65-69 probe5′-FAM-TAG TAG ATT “T” CAG AGA ACT TAA TAA GAG AAC TCAAGA CT (SEQ ID NO: 68) 69T Set 1 69T F1^(‡)5′-RTA TTT GCC ATA AAG AAR AAR RAY AAT AC (SEQ ID NO: 12) 69T F2^(‡)5′-RTA TTT GCC ATA AAG AAR AAR RAY AAC AC (SEQ ID NO: 13) 69T REV5′-GTA TGG TAA ATG CAG TAT ACT TCC T (SEQ ID NO: 14) ^(d)69Tmi5′-CCA RTA TTT GCC ATA AAG AAR AAR RAY AGT (SEQ ID NO: 15) 69T probe5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TYT CAR TTC CCT TA (SEQ ID NO: 9)69T Set 2 69T F1^(&)5′-RTA TTT GCC ATA AAG AAR AAR RAY AAT AC (SEQ ID NO: 12) 69T F25′-RTA TTT GCY ATA AAG AAR AAR GAY AGC AC (SEQ ID NO: 71) 69T REV^(#)5′-CCT GGT GTC TCA TTG TTT ATA CTA GGT (SEQ ID NO: 8) ^(d)69Tmi5′-CCA RTA TTT GCC ATA AAG AAR AAR RAY AGT (SEQ ID NO: 15) 65-69 probe5′-FAM-TAG TAG ATT “T” CAG AGA ACT TAA TAA GAG AAC TCAAGA CT (SEQ ID NO: 68) 70R Set 1 70R F15′-TRT TTG CCA TAA AGA AAA AAR AYA GTA MCA G (SEQ ID NO: 16) 70R F25′-TTG CCA TAA AGA AAA AAR ACA GTG ACA G (SEQ ID NO: 17) 70R F35′-TTG CCA TAA AGA AAA AAR ACA GYR ACA G (SEQ ID NO: 18) 70R F45′-GCC ATA AAG AAA AAA RAC RGT RAC GG (SEQ ID NO: 19) 70R REV5′-GTA TGG TAA ATG CAG TAT ACT TCC T (SEQ ID NO: 20) ^(d)70Rmi5′-AGT ATT TGC CAT AAA GAA AAA ARA CAG TAM TA (SEQ ID NO: 21) 70R probe5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TYT CAR TTC CCT TA (SEQ ID NO: 9)70R Set 2 70 F1^(£) 5′-AAA GTT AAA CAA TGG CCA TTG ACA G (SEQ ID NO: 2)70R REV1^(§) 5′-GTT CTC TRA AAT CTA YTA WTT TTC TCC CTC (SEQ ID NO: 72)70R REV2 5′-TTC TCT RAA ATC TAY TAW TTT TCT CCC CC (SEQ ID NO: 73)^(d)70mi 5′-AGT ATT TGC CAT AAA GAA AAA ARA CAG TAM TA (SEQ ID NO: 21)70R probe^(†) 5′-FAM-TTG GGC CTG AAA A“T”C CAT ACA ATA CTC CAG TAT TT(SEQ ID NO: 67) 70R Set3 70 F2^(£)5′-AGA RAT TTG TAC AGA RAT GGA AAA GGA AG (SEQ ID NO: 100) 70R REV1^(§)5′-GTT CTC TRA AAT CTA YTA WTT TTC TCC CTC (SEQ ID NO: 72) 70R REV25′-TTC TCT RAA ATC TAY TAW TTT TCT CCC CC (SEQ ID NO: 73) 70R probe^(†)5′-FAM-TTG GGC CTG AAA A“T”C CAT ACA ATA CTC CAG TAT TT (SEQ ID NO: 67)103N 103N F1 5′-TCC HGC AGG GTT AAA RAA GGA C (SEQ ID NO: 22) 103N F25′-TCC CKC WGG GTT AAR AAG GGA C (SEQ ID NO: 23) 103N F35′-CAT CCH GCA GGR TTA AAA AAG GGC (SEQ ID NO: 24) 103N F45′-CAT CCC GCA GGG TTA AAA VAG GAT (SEQ ID NO: 25) 103N REV5′-GTA TGG TAA ATG CAG TAT ACT TCC T (SEQ ID NO: 26) ^(d)103Nmi5′-CAT CCH GCA GGR CTA AAA AAG AA (SEQ ID NO: 27) 103N probe5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TYT CAR TTC CCT TA (SEQ ID NO: 9)181C 181C F1 5′-AAA ACA AAA YCC AGA MAT GRT TGG CTG (SEQ ID NO: 28)181C F2 5′-GAA AAC AAA AYC CAR AMA TRG TTG GHT G (SEQ ID NO: 29)181C REV 5′-CAG GAT GGA GTT CAT AAC CCA T (SEQ ID NO: 30) ^(d)181Cmi5′-TTY AGA AAA CAA AAY CCA GAM ATG RTT ATM T (SEQ ID NO: 31) 181C probe5′-FAM-TAG GAT CTG ACT TAG AAA “T”AG GRC AGC ATA GAR C (SEQ ID NO: 32)184V 184V F1 5′-AAA TCC ARA MMT ART TAT MTR TCA GCA CG (SEQ ID NO: 33)184V F2 5′-AAA TCC AGA MAT ART TAT CTR TCA GCA CG (SEQ ID NO: 34)184V F3 5′-AAA YCC ARA MAT ART TAT CTR YCA GCA TG (SEQ ID NO: 35)184V REV 5′-CAG GAT GGA GTT CAT AAC CCA T (SEQ ID NO: 36) ^(d)184Vmi5′-AAR CAA AAY CCA RAM ATA RTT ATC TRT CAA TAY (SEQ ID NO: 37)184V probe 5′-FAM-TAG GAT CTG ACT TAG AAA “T”AG GRC AGC ATA GAR C(SEQ ID NO: 32) 215 Y, F, S, C, D 215Y F15′-ASA RCA TCT GTK GAR RTG GGG RYT CTA (SEQ ID NO: 40) 215F F15′-ASA RCA TCT GTK GAR RTG GGG RYT CTT (SEQ ID NO: 41) 215S F15′-ARC ATC TGT KGA RGT GGG GRY TCT C (SEQ ID NO: 42) 215C F15′-ARC ATC TGT KGA RGT GGG GRY TCT G (SEQ ID NO: 43) 215D F15′-SAR CAT CTG TKG ARR TGG GGR YTC GA (SEQ ID NO: 44) 215REV5′-CTT CTG TAT GTC ATT GAC AGT CC (SEQ ID NO: 45) ^(d)215mi5′-SAA CAT CTG TTG ARG TGG GGR YTT (SEQ ID NO: 46) probe5′-FAM-TGG ACA GTA CAG CC“T” ATA RTG CTG CCA GA (SEQ ID NO: 47)215T Set 1 215T F1^(‡)5′-ACA TCT GTK GAR GIG GGG RYT CAC (SEQ ID NO: 38) 215T F2^(‡)5′-ASA AYA TCT GTT RAR GTG GGG RTT CAC (SEQ ID NO: 39) 215 REV5′-CTT CTG TAT GTC ATT GAC AGT CC (SEQ ID NO: 45) ^(d)215mi5′-SAA CAT CTG TTG ARG TGG GGR YTT (SEQ ID NO: 46) probe5′-FAM-TGG ACA GTA CAG CC“T” ATA RTG CIG CCA GA (SEQ ID NO: 47)215T Set 2 215T F1^(&)5′-AAC ATC TGT KGA RGT GGG GRY TCA C (SEQ ID NO: 74) 215T F25′-AAC ATY TGT TAA RGT GGG GRY TCA C (SEQ ID NO: 75) 215 REV5′-CTT CTG TAT GTC ATI GAC AGT CC (SEQ ID NO: 45) ^(d)215mi5′-SAA CAT CTG TTG ARG TGG GGR YTT (SEQ ID NO: 46) 215 probe15′-FAM-TAT GAA CTC CA“ T”C CTG ATA AAT GGA CAG TAC ARC (SEQ ID NO: 76)215 probe2 5′-FAM-TAT GAG CTC CA“ T”C CTG ATA AAT GGA CAG TRC(SEQ ID NO: 77) 215T Set 3 215T F3^(&)5′-CAA CAT YTG TTA ARG TGG GGR GAT AC (SEQ ID NO: 101) 215T F25′-AAC ATY TGT TAA RGT GGG GRY TCA C (SEQ ID NO: 75) 215 REV5′-CTT CTG TAT GTC ATT GAC AGT CC (SEQ ID NO: 45) ^(d)215mi5′-SAA CAT CTG TTG ARG TGG GGR YTT (SEQ ID NO: 46) 215 probe15′-FAM-TAT GAA CTC CA“ T”C CTG ATA AAT GGA CAG TAC ARC (SEQ ID NO: 76)215 probe2 5′-FAM-TAT GAG CTC CA“ T”C CTG ATA AAT GGA CAG TRC(SEQ ID NO: 77) Protease 30N 30N F15′-GCT YTA TTA GAY ACA GGR GCA GGT A (SEQ ID NO: 48) 30N F25′-GCT CTA TTM GAY ACA GGA GCW GGT A (SEQ ID NO: 49) 30N REV5′-TGG TAC AGT TTC AAT AGG ACT AAT GGG (SEQ ID NO: 50) ^(d) 30Nmi5′-CTY TAT TMG AYA CAG GRG CAG GTA (SEQ ID NO: 51) 30N probe5′-FAM-TAA RAC AGT ATG ATC AGR TAC CCA “T”AG AAA TCT GTG GAC-3'(SEQ ID NO: 52) 90M Set 1 90M F15′-CTG YCA ACR TAA TTG GAA GAA ATC CGA (SEQ ID NO: 53) 90M F25′-CTR CCA ACA TAA TTG GAA GAA AYC CGA (SEQ ID NO: 54) 90M F35′-CTR YCA ACR TAA TTG GAA GAA ATC CAA (SEQ ID NO: 55) 90 REV5′-CTT CTG TCA ATG GCC ATT GTT TAA C (SEQ ID NO: 56) ^(d) 90Mmi5′-CCT GYC AAC RTA ATT GGA AGA AAY CT (SEQ ID NO: 57) 90M probe5′-FAM-TGT ACC AGT AAA AT“T” AAA GCC AGG AAT GGA TGG (SEQ ID NO: 58)90M Set 2 90M F1 5′-TGY CAA CRT AAT TGG RAG RAA YCG GA (SEQ ID NO: 78)90M F2 5′-CTR YCA ACR TAA TTG GAA GRA ATK GGA (SEQ ID NO: 79) 90M F35′-CTR YCA ACR TAA TTG GAA GAA ATC CAA (SEQ ID NO: 55) 90M F45′-RYC AAC RTA ATT GGR AGA GAY CGG A (SEQ ID NO: 80) 90M REV5′-AAT GCT TTT ATT TTT TCT TCT GTC AAT GGC (SEQ ID NO: 81) ^(d) 90Mmi5′-CCT GYC AAC RTA ATT GGA AGA AAY CT (SEQ ID NO: 57) 90M probe5′-FAM-TAA ATT TTC CCA “T” TAG TCC TAT TGA AAC TGT ACC AGTAAA (SEQ ID NO: 82) Subtype C Reverse transcriptase 103N 103N F15′-CCC AGT AGG RTT AAA RAA GGA C (SEQ ID NO: 59) 103N F25′-CCC AKC RGG GTT RAA AGA GGA C (SEQ ID NO: 60) 103N F35′-CCC AGC AGG RTT AAA AVA GGA T (SEQ ID NO: 61) 103N REV5′-GTA TGG TAA ATG CAG TAT ACT TCC T (SEQ ID NO: 26) 103N probe5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TYT CAR TTC CCT TA (SEQ ID NO: 9)181C 181C F1 5′-GRA CAM AAA ATC CAG AAA TAG TYG CCT G (SEQ ID NO: 83)181C F2 5′-ACA MRA AAT CCA GAA ATA GTY GCT TG (SEQ ID NO: 84)181/184 REV 5′-CAG GAT GGA GTT CAT AAC CCA T (SEQ ID NO: 85)181/184 probe1 5′-FAM-TAG GAT CTG ATT “T” AGA AAT AGG GCA ACA TAGRAC (SEQ ID NO: 86) 181/184 probe2 5′-FAM-TAG GAT CTG ATT “T”AGA AAT AAA GCA ACA TAG RAC (SEQ ID NO: 87) 184V Set 1 184V F15′-AAA AYC CAG AMA TAR TYA TCT RYC AGC ATG (SEQ ID NO: 88) 184V F25′-MAA AAY CCA RAM ATA RTY ATM TRT CAG CAC G (SEQ ID NO: 89) 181/184 REV5′-CAG GAT GGA GTT CAT AAC CCA T (SEQ ID NO: 85) 181/184 probe15′-FAM-TAG GAT CTG ATT “T” AGA AAT AGG GCA ACA TAG RAC (SEQ ID NO: 86)181/184 probe2 5′-FAM-TAG GAT CTG ATT “T” AGA AAT AAA GCA ACA TAGRAC (SEQ ID NO: 87) 184V Set 2 184V F35′-AAA AYC CAG RAA TAR TYA TCT RTC AGC ATG (SEQ ID NO: 102) 184V F45′-AAY CCA GAM ATA RTY ATC TRT CAG CAC G (SEQ ID NO: 103) 184V F55′-AAA AYC CAG ARA TAR TYA TYT RTC AGC ATG (SEQ ID NO: 104) 181/184 REV5′-CAG GAT GGA GTT CAT AAC CCA T (SEQ ID NO: 85) 181/184 probe15′-FAM-TAG GAT CTG ATT “T” AGA AAT AGG GCA ACA TAG RAC (SEQ ID NO: 86)181/184 probe2 5′-FAM-TAG GAT CTG ATT “T” AGA AAT AAA GCA ACA TAGRAC (SEQ ID NO: 87) IUPAC codes: M = A or C; R = A or G; W = A or T; S =C or G; Y = C or T; K = G or T Notes: FAM = 6-carboxyfluorescein,however, any fluorophore may be used; the “”marks are where the quencheris placed ^(#)67 and 69 REV are the same as the comREV primer ^(§)Testperformed in reverse orientation where the reverse primers detect themutation ^(&)Test for the wildtype codon (absence of mutation) ^(†)Sameas the 41L probe ^(£)Same as the RT-PCR primer RTPF2

Simian Immunodeficiency virus (SIV) has strong clinical, pathological,virological and immunological analogies with HIV infection of humans.Infection of macaques with SIV provides a valuable model for exploringcrucial issues related to both the pathogenesis and prevention of HIVinfection. The model offers a unique setting for mutation detectiontesting, preclinical evaluation of drugs, vaccines and gene-therapiesagainst HIV, and can identify many virus and host determinants oflentiviral disease. As such, the present invention can be utilized inconjunction with SIV nucleotide sequences. Provided below are exemplarySIV sequences for use with the present invention. The SIVmac 65Rmutation-specific reaction can be compared against the total copy(common) reaction in the same way as described previously for HIVoligonucleotides.

Macaque SIV Reverse Transcriptase

Accession number: AY588945, M33262, AY599201, AY597209, M19499Exemplary Sequence (SEQ ID NO: 105) 1CCCATAGCTA AAGTAGAGCC TGTAAAAGTC GCCTTAAAGC CAGGAAAGGA TGGACCAAAA TTGAAGCAGT GGCCATTATC81AAAAGAAAAG ATAGTTGCAT TAAGAGAAAT CTGTGAAAAG ATGGAAAAGG ATGGTCAGTT GGAGGAAGCT CCCCCGACCA161ATCCATACAA CACCCCCACA TTTGCTATAA AGAAAAAGGA TAAGAACAAA TGGAGAATGC TGATAGATTT TAGGGAACTA241AATAGGGTCA CTCAGGACTT TACGGAAGTC CAATTAGGAA TACCACACCC TGCAGGACTA GCAAAAAGGA AAAGAATTAC321AGTACTGGAT ATAGGTGATG CATATTTCTC CATACCTCTA GATGAAGAAT TTAGGCAGTA CACTGCCTTT ACTTTACCAT401CAGTAAATAA TGCAGAGCCA GGAAAACGAT ACATTTATAA GGTTCTGCCT CAGGGATGGA AGGGGTCACC AGCCATCTTC481CAATACACTA TGAGACATGT GCTAGAACCC TTCAGGAAGG CAAATCCAGA TGTGACCTTA GTCCAGTATA TGGATGACAT561CTTAATAGCT AGTGACAGGA CAGACCTGGA ACATGACAGG GTAGTTTTAC AGTCAAAGGA ACTCTTGAAT AGCATAGGGT641TTTCTACCCC AGAAGAGAAA TTCCAAAAAG ATCCCCCATT TCAATGGATG GGGTACGAAT TGTGGCCAAC AAAATGGAAG721TTGCAAAAGA TAGAGTTGCC ACAAAGAGAG ACCTGGACAG TGAATGATAT ACAGAAGTTA GTAGGAGTAT TAAATTGGGC801AGCTCAAATT TATCCAGGTA TAAAAACCAA ACATCTCTGT AGGTTAATTA GAGGAAAAAT GACTCTAACA GAGGAAGTTC881AGTGGACTGA GATGGCAGAA GCAGAATATG AGGAAAATAA AATAATTCTC AGTCAGGAAC AAGAAGGATG TTATTACCAA961GAAGGCAAGC CATTAGAAGC CACGGTAATA AAGAGTCAGG ACAATCAGTG GTCTTATAAA ATTCACCAAG AAGACAAAAT1041ACTGAAAGTA GGAAAATTTG CAAAGATAAA GAATACACAT ACCAATGGAG TGAGACTATT AGCACATGTA ATACAGAAAA1121TAGGAAAGGA AGCAATAGTG ATCTGGGGAC AGGTCCCAAA ATTCCACTTA CCAGTTGAGA AGGATGTATG GGAACAGTGG1201TGGACAGACT ATTGGCAGGT AACCTGGATA CCGGAATGGG ATTTTATCTC AACACCACCG CTAGTAAGAT TAGTCTTCAA1281TCTAGTGAAG GACCCTATAG AGGGAGAAGA AACCTATTAT ACAGATGGAT CATGTAATAA ACAGTCAAAA GAAGGGAAAG1361CAGGATATAT CACAGATAGG GGCAAAGACA AAGTAAAAGT GTTAGAACAG ACTACTAATC AACAAGCAGA ATTGGAAGCA1441TTTCTCATGG CATTGACAGA CTCAGGGCCA AAGGCAAATA TTATAGTAGA TTCACAATAT GTTATGGGAA TAATAACAGG1521ATGCCCTACA GAATCAGAGA GCAGGCTAGT TAATCAAATA ATAGAAGAAA TGATTAAAAA GTCAGAAATT TATGTAGCAT1601GGGTACCAGC ACACAAAGGT ATAGGAGGAA ACCAAGAAAT AGACCACCTA GTTAGTCAAG GGATTAGACA AGTTCTCTTC1681TTGGAAAAGA TAGAGCCAGC ACAAGAAGAA CATGATAAAT ACCATAGTAA TGTAAAAGAA TTGGTATTCA AATTTGGATT1761ACCCAGAATA GTGGCCAGAC AGATAGTAGA CACCTGTGAT AAATGTCATC AGAAAGGAGA GGCTATACAT GGGCAGGCAA1841ATTCAGATCT AGGGACTTGG CAAATGGATT GTACCCATCT AGAGGGAAAA ATAATCATAG TTGCAGTACA TGTAGCTAGT1921GGATTCATAG AAGCAGAGGT AATTCCACAA GAGACAGGAA GACAGACAGC ACTATTTCTG TTAAAATTGG CAGGCAGATG2001GCCTATTACA CATCTACACA CAGATAATGG TGCTAACTTT GCTTCGCAAG AAGTAAAGAT GGTTGCATGG TGGGCAGGGA2081TAGAGCACAC CTTTGGGGTA CCATACAATC CACAGAGTCA GGGAGTAGTG GAAGCAATGA ATCACCACCT GAAAAATCAA2161ATAGATAGAA TCAGGGAACA AGCAAATTCA GTAGAAACCA TAGTATTAAT GGCAGTTCAT TGCATGAATT TTAAAAGAAG2241GGGAGGAATA GGGGATATGA CTCCAGCAGA AAGATTAATT AACATGATCA CTACAGAACA AGAGATACAA TTTCAACAAT2321CAAAAAACTC AAAATTTAAA AATTTTCGGG TCTATTACAG AGAAGGCAGA GATCAACTGT GGAAGGGACC CGGTGAGCTA2401TTGTGGAAAG GGGAAGGAGC AGTCATCTTA AAGGTAGGGA CAGACATTAA GGTAGTACCC AGAAGAAAGG CTAAAATTAT2481CAAAGATTAT GGAGGAGGAA AAGAGGTGGA TAGCAGTTCC CACATGGAGG ATACCGGAGA GGCTAGAGAG GTGGCATAGC2561CTCATAAAAT ATCTGAAATA TAAAACTAAA GATCTACAAA AGGTTTGCTA TGTGCCCCAT TTTAAGGTCG GATGGGCATG2641GTGGACCTGC AGCAGAGTAA TCTTCCCACT ACAGGAAGGA AGCCATTTAG AAGTACAAGG GTATTGGCAT TTGACACCAG2721AAAAAGGGTG GCTCAGTACT TATGCAGTGA GGATAACCTG GTACTCAAAG AACTTTTGGA CAGATGTAAC ACCAAACTAT2801GCAGACATTT TACTGCATAG CACTTATTTC CCTTGCTTTA CAGCGGGAGA AGTGAGAAGG GCCATCAGGG GAGAACAACT2881GCTGTCTTGC TGCAGGTTCC CGAGAGCTCA TAAGTACCAG GTACCAAGCC TACAGTACTT AGCACTGAAA GTAG

-   -   Genome Location: 1954 . . . 4907    -   Additional Similar Nucleotide Examples: Accession Numbers:        U65787        Protein: Accession Number: AAV65312

SIVmac

RT-PCR reaction: RTP F1 (SEQ ID NO: 106)5′-CAA AAG AAA AGA TAG TTG CAT TAA GAG AAA T RTP REV (SEQ ID NO: 107)5′-GCC ACA ATT CGT ACC CCA TCC AReverse Transcriptase

Total copy (SEQ ID NO: 108) com F1 5′-CAT ACA ACA CCC CCA CAT TTG CTA TA(SEQ ID NO: 109) com REV 5′-AGT CCT GCA GGG TGT GGT ATT C 65R(SEQ ID NO: 110) 65R F1 5′-ACT CCC ACA TTT GCY ATA GCG AG(SEQ ID NO: 111) com REV 5′-AGT CCT GCA GGG TGT GGT ATT C(SEQ ID NO: 112) probe 5′-FAM-TAG ATT TTA GGG AAC “T” AAA TAGGGT CAC TCA GGA C

Oligonucleotide Mixture Proportions

The following is a list of primers disclosed above with an example ofthe ratios/proportions of these primers that can be used to specificallyand sensitively detect the respective mutations.

Subtype B

Reverse

transcriptase

41L 41L F2 (35%) (SEQ ID NO: 63) 41L F5 (10%) (SEQ ID NO: 96) 41L F6(32%) (SEQ ID NO: 97) 41L F3 (13%) (SEQ ID NO: 64) 41L F4 (10%) (SEQ IDNO: 65) 67N 67N F2 (60%) (SEQ ID NO: 69) 67N F3 (40%) (SEQ ID NO: 70)69T Set 1 69T F1^(‡) (60%) (SEQ ID NO: 12) 69T F2^(‡) (40%) (SEQ ID NO:13) 69T Set 2 69T F1^(‡) (60%) (SEQ ID NO: 12) 69T F2^(‡) (40%) (SEQ IDNO: 71) 70R Set 1 70F1 (40%) (SEQ ID NO: 16) 70F2 (12%) (SEQ ID NO: 17)70F3 (10%) (SEQ ID NO: 18) 70F4 (38%) (SEQ ID NO: 19) 70R Set 3 70RREV1(70%) (SEQ ID NO: 72) 70R REV2(30%) (SEQ ID NO: 73) 103N 103F1 (40%)(SEQ ID NO: 22) 103F2 (12%) (SEQ ID NO: 23) 103F3 (10%) (SEQ ID NO: 24)103F4 (38%) (SEQ ID NO: 25) 181C 181F1 (76%) (SEQ ID NO: 28) 181F2 (34%)(SEQ ID NO: 29) 184V 184F1 (50%) (SEQ ID NO: 33) 184F2 (15%) (SEQ ID NO:34) 184F3 (35%) (SEQ ID NO: 35) 215T Set 1 215T F1 (70%) (SEQ ID NO: 38)215T F2 (30%) (SEQ ID NO: 39) 215T Set 3 215T F3^(‡) (70%) (SEQ ID NO:101) 215T F2^(‡) (30%) (SEQ ID NO: 75)Protease

30N 30N F1 (70%) (SEQ ID NO: 48) 30N F2 (30%) (SEQ ID NO: 49) 90M 90M F1(36%) (SEQ ID NO: 78) 90M F2 (33%) (SEQ ID NO: 79) 90M F3 (16%) (SEQ IDNO: 55) 90M F4 (15%) (SEQ ID NO: 80)Subtype CReversetranscriptase

103N 103CF1 (47%) (SEQ ID NO: 59) 103CF2 (33%) (SEQ ID NO: 60) 103CF3(20%) (SEQ ID NO: 61) 181C 181C F1 (72.5%) (SEQ ID NO: 83) 181C F2(27.5%) (SEQ ID NO: 84) 184V 184V F3 (35%) (SEQ ID NO: 102) 184V F4(40%) (SEQ ID NO: 103) 184V F5 (25%) (SEQ ID NO: 104)

103N and 184V Assay Sensitivity with Virus Mixtures

When tested against plasmid clones the mixed-primer assay for 103N wasable to distinguish as little as 0.04% 103N in within a wild typebackground. The assay for 184V yielded discernable CTs for 184V plasmidswhen comprising as little as 0.2% of the population (FIG. 2A).

103N and 181C Assay Performance on Clinical Samples

To determine the overall assay performance on clinical specimens andestablish the assay cutoff values, the data for the knownpatient-derived wild types and the 103N and 184V mutants were collated.An example of the performance of the 184V assay on a clinical specimenthat carried the mutation and on a sample that had only wild type virusis shown in FIG. 3. The resulting distribution of collated ΔCTs revealedthe best placement for the 103N cutoff to be a ΔCT of 12 and the 184Vcutoff to be at a ΔCT of 8.5. That is, a ΔCT below these values isscored positive for the respective mutation, while a ΔCT above is scoredas having only wild type (FIG. 4). As a group, the ΔCTs of the specimensdocumented to have mutations were significantly different from the ΔCTsof the wild type samples and samples possessing other mutations(P<0.001) (Table I). The 184V assay did not detect this mutation in anyof the 77 documented wild type samples. However, with the 103N assay, 1wild type sample scored positive (ΔCT of 10.6) for the mutation. The103N discordant result might be signifying a very low level (<5%)naturally occurring polymorphism. The assay for the 103N mutation wasable to detect the mutation in all 23 samples documented to have themutation. The 184V assay was unable to detect the mutation in one (ΔCTof 9.8) of the 36 specimens known to have the mutation, yielding anassay sensitivity of 97.2%. This outlier was obtained from atreatment-experienced person having a mixed virus population with fivepolymorphisms in the primer binding site.

Using the 12.0 ΔCT cutoff for the 103N assay, none of 69 specimensdocumented to have mutations other than 103N scored positive. With the8.5 ΔCT cutoff for 184V, one specimen previously determined to benegative for 184V scored positive (ΔCT of 7.1), giving the assay anoverall specificity of 98.6%. This discordant sample was from achronically infected, treatment-naïve person infected with viruscarrying 41L and 215D RT mutations.

TABLE I ΔCT Measures for Each Group of Clinical Samples Mean ΔCT MedianΔCT 103N: Early wildtype 17.0 16.7 Naïve wildtype 18.8 17.1 Othermutants 19.5 18.6 103N mutants 5.8 5.5 184V: Early wildtype 10.9 10.2Naïve wildtype 12.5 11.1 Other mutants 12.8 11.7 184V mutants 5.0 5.1

Performance of the 70R, 90M and 67N Assays on Transmitted Drug-ResistantViruses

The subtype B 70R assay cutoff=9.0 cycles, 90M assay cutoff=10.0 cycles,and 67N assay cutoff=9.0 cycles.

To reduce both the chance of false-positive results and the detection ofnaturally-occurring resistance-associated polymorphisms, assay cutoffsof 0.2-0.5% mutant virus were used for screening purposes. Thesensitivities and specificities of the assays on genotyped clinicalsamples carrying the mutations of interest were found to range between95-99%. Real-time PCR screening of the 147 transmitted HIV-1 carryingresistance-related mutations detected additional mutations that expandedthe spectrum of drugs to which the viruses were resistant. The addedmutants increased the prevalence of 90M from 8% to 10% (+25%), of 184Vfrom 10% to 12% (+20%), of 70R from 9% to 14% (+56%), and of 67N from 7%to 12% (+71%).

HIV-1 Subtype C 103N and 181C Findings from a Study Examining theEmergence of Resistance in Women Receiving Intrapartum Single-DoseNevirapine

The subtype C HIV-1 103N assay cutoff=11.0 cycles, and the 181C assaycutoff=9.0 cycles.

The 103N real-time assay confirmed the absence of detectable 103N in all50 pre-NVP baseline samples (ΔCT range of 12.0-23.0 cycles, meanΔCT=15.9 cycles) (FIG. 5). The assay successfully detected 103N in all10 post-NVP positive control

specimens (ΔCT range of 2.8-9.8 cycles, mean ΔCT=6.6 cycles). Of the 40post-NVP specimens that had no detectable NVP-related mutations bysequencing, the real-time

PCR assay found 16 (40%) were positive for 103N (ΔCT range of 6.9-10.6cycles, mean ΔCT=8.9 cycles) (FIG. 3, table 1). The ΔCT values for thenew-found 103N—

positive specimens were significantly lower than the pre-NVP specimens(ΔΔCT) (paired

FIG. 5 shows the real-time PCR analysis of the 103N mutation in pre-NVPand post-NVP plasma samples. Seq +/−, sequence analysispositive/negative for 103N; PCR+/−, real-time PCR positive/negative for103N. A ΔCT value at or above the cutoff indicates 103N is not detected,a value below indicates the presence of 103N. T-test, p<0.0001,range=−(3.2-8.3) cycles, mean ΔΔCT=−6.0 cycles). In contrast, nosignificant difference was seen between the pre-NVP specimens and thenegative post-NVP specimens (p=0.61). The resistant variants wereidentified in samples collected throughout the entire 36-week postpartumperiod.

The present real-time PCR primer-mix point mutation assay for the HIV-1103N and 184V RT mutation were able to detect as little as 0.04% and0.2% mutant virus, respectively, in HIV-1 plasmid dilutions. The primerdesigns were robust and worked well with the very high sequencevariability in the clinical specimens examined. The ΔCTs of themutation-positive specimens formed a distinct cluster from the wild typesamples and samples with other mutations. These assays have shownacceptable performance on 282 samples of plasma-derived HIV-1, providinga sensitivity of 97.2-100% and a specificity of 98.6%.

The benefits of real-time PCR-based testing include the following: 1)The real-time reaction requires a one-step setup, decreasing thepotential for user error; 2) High throughput: reactions performed in96-well plate allowing up to 40 patient samples per plate with resultsin <3 hrs; 3) The use of primer mixtures can decrease the frequency of“no calls” often seen with other point mutation assays as a result ofadjacent polymorphic mismatches; 4) This amplification-based technologyis much more sensitive than conventional sequencing, and can be usefulas both a primary screening tool and for post treatment evaluation; 5)This technology is currently used in public health lab settings and maybe transferred to locations where current genotyping is cost-prohibited;and 6) Real-time PCR is a powerful tool that can garner simultaneousvirologic measures (e.g., virus load and resistance load).

REFERENCES

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Morespecifically, they are incorporated for the teaching recited in thecontext in which they are disclosed herein.

-   1. Low-Frequency NNRTI-resistant variants contribute to failure of    efavirenz-containing regimens. J. Mellors, S. Palmer, D. Nissley, M.    Kearney, E. Halvas, C. Bixby, L. Demeter, S. Eshelman, K.    Bennett, S. Hart, F. Vaida, M. Wantman, J. Coffin, and S. Hammer.    Abstract from the 11^(th) CROI, San Francisco, Calif. (Feb. 9-11,    2004).-   2. The epidemiology of antiretroviral drug resistance among    drug-naive HIV-1 infected persons in 10 U.S. cities. H. S.    Weinstock, I. Zaidi, W. Heneine, D. Bennett, J. G.    Garcia-Lerma, J. M. Douglas, Jr., M. LaLota, G. Dickinson, S.    Schwarcz, L. Torian, D. Wendell, S. Paul, G. A. Goza, J. Ruiz, B.    Boyett, and J. E. Kaplan. (JID in press)-   3. Changes in human immunodeficiency virus type 1 populations after    treatment interruption in patients failing antiretroviral therapy.    Hance A J, Lemiale V, Izopet J, Lecossier D, Joly V, Massip P,    Mammano F, Descamps D, Brun-Vezinet F, Clavel F. J Virol 2001 July;    75(14):6410-7.-   4. New real-time PCR Assay quantifies K103N NNRTI-resistant variant    at a frequency as low as 0.01%. S Palmer, V. Boltz, F. Maldarelli, E    Halvas, J Mellors and J. Coffin. Abstract from the Third HIV DRP    Symposium: Antiviral Drug Resistance, Chantilly, Va. (Dec. 8-11,    2002)

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed:
 1. An oligonucleotide primer composition for thedetection of at least one mutation relating to a HIV-1 protein ofreverse transcriptase by hybridizing with a nucleic acid encoding theHIV-1 protein of reverse transcriptase, wherein the at least onemutation is selected from the group consisting of 184V, comprising: aprimer selective for the 184V mutation, the primer selected from thegroup of: SEQ ID NOs: 88 and 89
 44. 2. An oligonucleotide primercomposition for the detection of at least one mutation relating to aHIV-1 protein of reverse transcriptase by hybridizing with a nucleicacid encoding the HIV-1 protein of reverse transcriptase, wherein the atleast one mutation is 65R, comprising: a primer selective for the 65Rmutation, the primer having SEQ ID NO:
 10. 3. A group of oligonucleotideprimers for the detection of two mutations relating to a HIV-1 proteinof reverse transcriptase by hybridizing with a nucleic acid encoding theHIV-1 protein of reverse transcriptase, wherein the two mutations are65R and 184V, comprising: a primer selective for the 184V mutation, theprimer selected from the group of: SEQ ID NOs: 88 and 89; and a primerselective for the 65R mutation, the primer having SEQ ID NO: 10.